Conserved neisserial antigens

ABSTRACT

To ensure maximum cross-strain recognition and reactivity, regions of proteins that are conserved between different Neisserial species, serogroups and strains can be used. The invention provides proteins which comprise stretches of amino acid sequence that are shared across the majority of  Neisseria , particularly  N. meningitidis  and  N. gonorrhoeae.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a Divisional of U.S. patent application Ser. No.09/980,602, filed on Sep. 20, 2002, entitled CONSERVED NEISSERIALANTIGENS, which is the National Stage of International Application No.PCT/IB00/00642, filed Apr. 28, 2000, which claims the benefit of UnitedKingdom Application No. 0005728.1, titled CONSERVED NEISSERIAL ANTIGENS,filed Mar. 9, 2000; and the benefit of United Kingdom Application No.9910168.5, titled CONSERVED NEISSERIAL ANTIGENS, filed Apr. 30, 1999;all of which are incorporated herein by reference in their entireties.

FIELD OF THE INVENTION

This invention relates to conserved antigens from the Neisseriabacteria.

BACKGROUND ART

Neisseria meningitidis and Neisseria gonorrhoeae are non-motile, gramnegative diplococci that are pathogenic in humans.

Based on the organism's capsular polysaccharide, 12 serogroups of N.meningitidis have been identified. Group A is the pathogen most oftenimplicated in epidemic disease in sub-Saharan Africa. Serogroups B and Care responsible for the vast majority of cases in the United States andin most developed countries. Serogroups W135 and Y are responsible forthe rest of the cases in the United States and developed countries.

The meningococcal vaccine currently in use is a tetravalentpolysaccharide vaccine composed of serogroups A, C, Y and W135. Thisapproach cannot be used for Meningococcus B, however, because the menBcapsular polysaccharide is a polymer of α(2-8)-linked N-acetylneuraminic acid that is also present in mammalian tissue. One approachto a menB vaccine uses mixtures of outer membrane proteins (OMPs) Toovercome the antigenic variability, multivalent vaccines containing upto nine different porins have been constructed [eg. Poolman (1992)Development of a meningococcal vaccine. Infect. Agents Dis. 4:13-28].Additional proteins to be used in outer membrane vaccines have been theopa and opc proteins, but none of these approaches have been able toovercome the antigenic variability [eg. Ala'Aldeen & Borriello (1996)The meningococcal transferrin-binding proteins 1 and 2 are both surfaceexposed and generate bactericidal antibodies capable of killinghomologous and heterologous strains. Vaccine 14(1):49-53].

A large number of Neisserial protein and nucleotide sequences aredisclosed in WO99/24578, WO99/36544, WO99/57280 and WO00/22430. Thecontents of these four applications are incorporated herein byreference. Comprehensive sequence data from strain MC58 is disclosed inTettelin et al. [Science (2000) 287:1809-1815], the contents of whichare also incorporated herein by reference.

DESCRIPTION OF THE INVENTION

To ensure maximum cross-strain recognition and reactivity, regions ofproteins that are conserved between different Neisserial species,serogroups and strains can be used. The invention therefore providesproteins which comprise stretches of amino acid sequence that are sharedacross the majority of Neisseria, particularly N. meningitidis and N.gonorrhoeae.

The invention provides a protein comprising a fragment of a Neisserialprotein, wherein said fragment consists of n consecutive conserved aminoacids, with the proviso that the invention does not include within itsscope full-length Neisserial proteins. Depending on the particularprotein, n is 7 or more (eg. 8, 10, 12, 14, 16, 18, 20 or more). Thefragment preferably comprises an antigenic or immunogenic region of theNeisserial protein.

A “conserved” amino acid is one that is present in a particularNeisserial protein in at least x % of Neisseria. The value of x may be50% or more eg. 66%, 75%, 80%, 90%, 95% or even 100% (ie. the amino acidis found in the protein in question in all Neisseria).

In order to determine whether an amino acid is “conserved” in aparticular Neisserial protein, it is necessary to compare that aminoacid residue in the sequences of the protein in question from aplurality of different Neisseria (a “reference population”). Thereference population may include a number of different Neisseria species(preferably N. meningitidis and N. gonorrhoeae) or may include a singlespecies. The reference population may include a number of differentserogroups of a particular species (such as the A, B, C, W135, X, Y, Zand 29E serogroups of N. meningitidis) or a single serogroup. Thereference population may also include a number of different strains froma particular serogroup (such as the NG6/88, BZ198, NG3/88, 297-0, BZ147,BZ169, 528, BZ133, NGE31, NGH38, NGH15, BZ232, BZ83, and 44/76 strainsof N. meningitidis B). A preferred reference population consists of the5 most common strains of N. meningitidis and/or the 5 most commonstrains of N. gonorrhoeae.

The reference population preferably comprises k strains taken from kdifferent branches of a suitable phylogenetic tree, such as thosedisclosed in (a) Ni et al. (1992) Epidemiol Infect 109:227-239 (b) Wolffet al. (1992) Nucleic Acids Res 20:4657 (c) Bygraves & Maiden (1992) J.Gen. Microbiol. 138:523-531 (d) Caugant et al. (1987) J. Bacteriol.69:2781-2792. Another phylogenetic tree that can be used is shown inFIG. 8 herein, and another in FIG. 9 b.

It will be appreciated that a particular species, serogroup or strainshould only be included in the reference population if it encodes theprotein in which the amino acid in question is located. In the case ofamino acids within ORF40 described below, for instance, the referencepopulation should not include N. gonorrhoeae because this species doesnot contain ORF40.

For proteins found in both N. meningitidis and N. gonorrhoeae,therefore, a preferred reference population comprises:

-   -   N. meningitidis A, strain Z2491    -   N. meningitidis B, strains NG6/88    -   N. meningitidis W, strains A22    -   N. gonorrhoeae, strain Ng F62

These are described in (a) Seiler A. et al. (1996) Mol. Microbiol.19(4):841-856 (b) Maiden et al. (1998) Proc. Natl. Acad. Sci. USA95:3140-3145 (c) Virji et al. (1992) Mol. Microbiol. 6:1271-1279 (d)Dempsey et al. (1991) J. Bacteriol. 173:5476-5486.

For proteins found only in N. meningitidis, however, a preferredreference population comprises:

-   -   N. meningitidis A, strain Z2491    -   N. meningitidis B, strains NG6/88    -   N. meningitidis W, strains A22

Amino acid sequences of different Neissieriae can easily be comparedusing computers. This will typically involve the alignment of a numberof sequences using an algorithm such as CLUSTAL [Thompson et al. (1994)Nucleic Acids Res 22:4673-4680; Trends Biochem Sci (1998) 23:403-405]or, preferably, PILEUP [part of the GCG Wisconsin package, preferablyversion 9.0].

Conserved amino acids are readily apparent in a multiple sequencealignment—at the amino acid position in question a majority of thealigned sequences will contain a particular amino acid. Conserved aminoacids can be made more visually apparent by using a program such asBOXSHADE [available, for instance, at the NIH on-line], PRETTYBOX [GCGWisconsin, version 10] or JALVIEW [available on-line at EBI].

The protein preferably comprises a fragment of one of the proteinsdisclosed in WO99/24578, WO99/36544, WO99/57280 or WO0/22430, or of oneof the 2158 ORFs disclosed in Tettelin et al. [Science (2000)287:1809-1815]. More particularly, it preferably comprises a fragment ofone or more of ORF4, ORF40, ORF46, protein 225, protein 235, protein287, protein 519, protein 726, protein 919 and protein 953 disclosedtherein (see examples herein). Typically, the protein of the inventionwill not comprise a protein sequence explicitly disclosed in WO99/24578,WO99/36544, WO99/57280, WO00/22430, or Tettelin et al.

The invention also provides a protein comprising one of the sequencesshown in the Figures.

The proteins of the invention can, of course, be prepared by variousmeans (eg. recombinant expression, native expression, purification fromcell culture, chemical synthesis etc.) and in various forms (eg. native,fusions etc.). They are preferably prepared in substantially pure form(ie. substantially free from other Neisserial or host cell proteins)

According to a further aspect, the invention provides antibodies whichbind to these proteins. These may be polyclonal or monoclonal and may beproduced by any suitable means.

According to a further aspect, the invention provides nucleic acidencoding the proteins of the invention. It should also be appreciatedthat the invention provides nucleic acid comprising sequencescomplementary to these (eg. for antisense or probing purposes).

Furthermore, the invention provides nucleic acid which can hybridise tothe N. meningitidis nucleic acid disclosed in the examples, preferablyunder “high stringency” conditions (eg. 65° C. in a 0.1×SSC, 0.5% SDSsolution).

Nucleic acid according to the invention can, of course, be prepared inmany ways (eg. by chemical synthesis, from genomic or cDNA libraries,from the organism itself etc.) and can take various forms (eg. singlestranded, double stranded, vectors, probes etc.).

In addition, the term “nucleic acid” includes DNA and RNA, and alsotheir analogues, such as those containing modified backbones, and alsopeptide nucleic acids (PNA) etc.

According to a further aspect, the invention provides vectors comprisingnucleotide sequences of the invention (eg. expression vectors) and hostcells transformed with them.

According to a further aspect, the invention provides compositionscomprising protein, antibody, and/or nucleic acid according to theinvention. These compositions may be suitable as vaccines, for instance,or as diagnostic reagents, or as immunogenic compositions.

The invention also provides nucleic acid, protein, or antibody accordingto the invention for use as medicaments (eg. as vaccines) or asdiagnostic reagents. It also provides the use of nucleic acid, protein,or antibody according to the invention in the manufacture of: (i) amedicament for treating or preventing infection due to Neisserialbacteria; (ii) a diagnostic reagent for detecting the presence ofNeisserial bacteria or of antibodies raised against Neisserial bacteria;and/or (iii) a reagent which can raise antibodies against Neisserialbacteria. The use is preferably applicable to all species of Neisseria.

Where a Neisserial protein contains more than q % conserved amino acids,the invention provides the use of the Neisserial protein, or a fragmentthereof, as a non-strain-specific protein that exhibits cross-reactivitybetween many species, serogroups and strains. The value of q may be 50%,60%, 75%, 80%, 90%, 95% or even 100%.

The invention also provides a method of treating a patient, comprisingadministering to the patient a therapeutically effective amount ofnucleic acid, protein, and/or antibody according to the invention.

According to further aspects, the invention provides various processes.

A process for producing proteins of the invention is provided,comprising the step of culturing a host cell according to the inventionunder conditions which induce protein expression.

A process for producing protein or nucleic acid of the invention isprovided, wherein the protein or nucleic acid is synthesised in part orin whole using chemical means.

A process for detecting polynucleotides of the invention is provided,comprising the steps of: (a) contacting a nucleic probe according to theinvention with a biological sample under hybridizing conditions to formduplexes; and (b) detecting said duplexes.

A process for detecting proteins of the invention is provided,comprising the steps of: (a) contacting an antibody according to theinvention with a biological sample under conditions suitable for theformation of an antibody-antigen complexes; and (b) detecting saidcomplexes.

A summary of standard techniques and procedures which may be employed inorder to perform the invention (eg. to utilise the disclosed sequencesfor vaccination or diagnostic purposes) follows. This summary is not alimitation on the invention but, rather, gives examples that may beused, but are not required.

General

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA, and immunology, which are within the skill of the art.Such techniques are explained fully in the literature eg. SambrookMolecular Cloning; A Laboratory Manual, Second Edition (1989); DNACloning, Volumes I and ii (D. N Glover ed. 1985); OligonucleotideSynthesis (M. J. Gait ed, 1984); Nucleic Acid Hybridization (B. D. Hames& S. J. Higgins eds. 1984); Transcription and Translation (B. D. Hames &S. J. Higgins eds. 1984); Animal Cell Culture (R. I. Freshney ed. 1986);Immobilized Cells and Enzymes (IRL Press, 1986); B. Perbal, A PracticalGuide to Molecular Cloning (1984); the Methods in Enzymology series(Academic Press, Inc.), especially volumes 154 & 155; Gene TransferVectors for Mammalian Cells (J. H. Miller and M. P. Calos eds. 1987,Cold Spring Harbor Laboratory); Mayer and Walker, eds. (1987),Immunochemical Methods in Cell and Molecular Biology (Academic Press,London); Scopes, (1987) Protein Purification: Principles and Practice,Second Edition (Springer-Verlag, N.Y.), and Handbook of ExperimentalImmunology, Volumes I-IV (D. M. Weir and C. C. Blackwell eds 1986).

Standard abbreviations for nucleotides and amino acids are used in thisspecification.

All publications, patents, and patent applications cited herein areincorporated in full by reference. In particular, the contents ofinternational patent applications WO99/24578, WO99/36544, WO99/57280 andWO00/22430 are incorporated herein.

DEFINITIONS

A composition containing X is “substantially free of” Y when at least85% by weight of the total X+Y in the composition is X. Preferably, Xcomprises at least about 90% by weight of the total of X+Y in thecomposition, more preferably at least about 95% or even 99% by weight.

The term “comprising” means “including” as well as “consisting” eg. acomposition “comprising” X may consist exclusively of X or may includesomething additional to X, such as X+Y.

The term “heterologous” refers to two biological components that are notfound together in nature. The components may be host cells, genes, orregulatory regions, such as promoters. Although the heterologouscomponents are not found together in nature, they can function together,as when a promoter heterologous to a gene is operably linked to thegene. Another example is where a Neisserial sequence is heterologous toa mouse host cell. A further examples would be two epitopes from thesame or different proteins which have been assembled in a single proteinin an arrangement not found in nature.

An “origin of replication” is a polynucleotide sequence that initiatesand regulates replication of polynucleotides, such as an expressionvector. The origin of replication behaves as an autonomous unit ofpolynucleotide replication within a cell, capable of replication underits own control. An origin of replication may be needed for a vector toreplicate in a particular host cell. With certain origins ofreplication, an expression vector can be reproduced at a high copynumber in the presence of the appropriate proteins within the cell.Examples of origins are the autonomously replicating sequences, whichare effective in yeast; and the viral T-antigen, effective in COS-7cells.

A “mutant” sequence is defined as DNA, RNA or amino acid sequencediffering from but having sequence identity with the native or disclosedsequence. Depending on the particular sequence, the degree of sequenceidentity between the native or disclosed sequence and the mutantsequence is preferably greater than 50% (eg. 60%, 70%, 80%, 90%, 95%,99% or more, calculated using the Smith-Waterman algorithm as describedabove). As used herein, an “allelic variant” of a nucleic acid molecule,or region, for which nucleic acid sequence is provided herein is anucleic acid molecule, or region, that occurs essentially at the samelocus in the genome of another or second isolate, and that, due tonatural variation caused by, for example, mutation or recombination, hasa similar but not identical nucleic acid sequence. A coding regionallelic variant typically encodes a protein having similar activity tothat of the protein encoded by the gene to which it is being compared.An allelic variant can also comprise an alteration in the 5′ or 3′untranslated regions of the gene, such as in regulatory control regions(eg. see U.S. Pat. No. 5,753,235).

Expression Systems

The Neisserial nucleotide sequences can be expressed in a variety ofdifferent expression systems; for example those used with mammaliancells, baculoviruses, plants, bacteria, and yeast.

i. Mammalian Systems

Mammalian expression systems are known in the art. A mammalian promoteris any DNA sequence capable of binding mammalian RNA polymerase andinitiating the downstream (3′) transcription of a coding sequence (eg.structural gene) into mRNA. A promoter will have a transcriptioninitiating region, which is usually placed proximal to the 5′ end of thecoding sequence, and a TATA box, usually located 25-30 base pairs (bp)upstream of the transcription initiation site. The TATA box is thoughtto direct RNA polymerase II to begin RNA synthesis at the correct site.A mammalian promoter will also contain an upstream promoter element,usually located within 100 to 200 bp upstream of the TATA box. Anupstream promoter element determines the rate at which transcription isinitiated and can act in either orientation [Sambrook et al. (1989)“Expression of Cloned Genes in Mammalian Cells.” In Molecular Cloning: ALaboratory Manual, 2nd ed.].

Mammalian viral genes are often highly expressed and have a broad hostrange; therefore sequences encoding mammalian viral genes provideparticularly useful promoter sequences. Examples include the SV40 earlypromoter, mouse mammary tumor virus LTR promoter, adenovirus major latepromoter (Ad MLP), and herpes simplex virus promoter. In addition,sequences derived from non-viral genes, such as the murinemetallotheionein gene, also provide useful promoter sequences.Expression may be either constitutive or regulated (inducible),depending on the promoter can be induced with glucocorticoid inhormone-responsive cells.

The presence of an enhancer element (enhancer), combined with thepromoter elements described above, will usually increase expressionlevels. An enhancer is a regulatory DNA sequence that can stimulatetranscription up to 1000-fold when linked to homologous or heterologouspromoters, with synthesis beginning at the normal RNA start site.Enhancers are also active when they are placed upstream or downstreamfrom the transcription initiation site, in either normal or flippedorientation, or at a distance of more than 1000 nucleotides from thepromoter [Maniatis et al. (1987) Science 236:1237; Alberts et al. (1989)Molecular Biology of the Cell, 2nd ed.]. Enhancer elements derived fromviruses may be particularly useful, because they usually have a broaderhost range. Examples include the SV40 early gene enhancer [Dijkema et al(1985) EMBO J. 4:761] and the enhancer/promoters derived from the longterminal repeat (LTR) of the Rous Sarcoma Virus [Gorman et al. (1982b)Proc. Natl. Acad. Sci. 79:6777] and from human cytomegalovirus [Boshartet al. (1985) Cell 41:521]. Additionally, some enhancers are regulatableand become active only in the presence of an inducer, such as a hormoneor metal ion [Sassone-Corsi and Borelli (1986) Trends Genet. 2:215;Maniatis et al. (1987) Science 236:1237].

A DNA molecule may be expressed intracellularly in mammalian cells. Apromoter sequence may be directly linked with the DNA molecule, in whichcase the first amino acid at the N-terminus of the recombinant proteinwill always be a methionine, which is encoded by the ATG start codon. Ifdesired, the N-terminus may be cleaved from the protein by in vitroincubation with cyanogen bromide.

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provides forsecretion of the foreign protein in mammalian cells. Preferably, thereare processing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell. Theadenovirus triparite leader is an example of a leader sequence thatprovides for secretion of a foreign protein in mammalian cells.

Usually, transcription termination and polyadenylation sequencesrecognized by mammalian cells are regulatory regions located 3′ to thetranslation stop codon and thus, together with the promoter elements,flank the coding sequence. The 3′ terminus of the mature mRNA is formedby site-specific post-transcriptional cleavage and polyadenylation[Birnstiel et al. (1985) Cell 41:349; Proudfoot and Whitelaw (1988)“Termination and 3′ end processing of eukaryotic RNA. In Transcriptionand splicing (ed. B. D. Hames and D. M. Glover); Proudfoot (1989) TrendsBiochem. Sci. 14:105]. These sequences direct the transcription of anmRNA which can be translated into the polypeptide encoded by the DNA.Examples of transcription terminator/polyadenylation signals includethose derived from SV40 [Sambrook et al (1989) “Expression of clonedgenes in cultured mammalian cells.” In Molecular Cloning. A LaboratoryManual].

Usually, the above described components, comprising a promoter,polyadenylation signal, and transcription termination sequence are puttogether into expression constructs. Enhancers, introns with functionalsplice donor and acceptor sites, and leader sequences may also beincluded in an expression construct, if desired. Expression constructsare often maintained in a replicon, such as an extrachromosomal element(eg. plasmids) capable of stable maintenance in a host, such asmammalian cells or bacteria. Mammalian replication systems include thosederived from animal viruses, which require trans-acting factors toreplicate. For example, plasmids containing the replication systems ofpapovaviruses, such as SV40 [Gluzman (1981) Cell 23:175] orpolyomavirus, replicate to extremely high copy number in the presence ofthe appropriate viral T antigen. Additional examples of mammalianreplicons include those derived from bovine papillomavirus andEpstein-Barr virus. Additionally, the replicon may have two replicationsystems, thus allowing it to be maintained, for example, in mammaliancells for expression and in a prokaryotic host for cloning andamplification. Examples of such mammalian-bacteria shuttle vectorsinclude pMT2 [Kaufman et al. (1989) Mol. Cell. Biol. 9:946] and pHEBO[Shimizu et al. (1986) Mol. Cell. Biol. 6:1074].

The transformation procedure used depends upon the host to betransformed. Methods for introduction of heterologous polynucleotidesinto mammalian cells are known in the art and include dextran-mediatedtransfection, calcium phosphate precipitation, polybrene mediatedtransfection, protoplast fusion, electroporation, encapsulation of thepolynucleotide(s) in liposomes, and direct microinjection of the DNAinto nuclei.

Mammalian cell lines available as hosts for expression are known in theart and include many immortalized cell lines available from the AmericanType Culture Collection (ATCC), including but not limited to, Chinesehamster ovary (CHO) cells, HeLa cells, baby hamster kidney (BHK) cells,monkey kidney cells (COS), human hepatocellular carcinoma cells (eg. HepG2), and a number of other cell lines.

ii. Baculovirus Systems

The polynucleotide encoding the protein can also be inserted into asuitable insect expression vector, and is operably linked to the controlelements within that vector. Vector construction employs techniqueswhich are known in the art. Generally, the components of the expressionsystem include a transfer vector, usually a bacterial plasmid, whichcontains both a fragment of the baculovirus genome, and a convenientrestriction site for insertion of the heterologous gene or genes to beexpressed; a wild type baculovirus with a sequence homologous to thebaculovirus-specific fragment in the transfer vector (this allows forthe homologous recombination of the heterologous gene in to thebaculovirus genome); and appropriate insect host cells and growth media.

After inserting the DNA sequence encoding the protein into the transfervector, the vector and the wild type viral genome are transfected intoan insect host cell where the vector and viral genome are allowed torecombine. The packaged recombinant virus is expressed and recombinantplaques are identified and purified. Materials and methods forbaculovirus/insect cell expression systems are commercially available inkit form from, inter alia, Invitrogen, San Diego Calif. (“MaxBac” kit).These techniques are generally known to those skilled in the art andfully described in Summers and Smith, Texas Agricultural ExperimentStation Bulletin No. 1555 (1987) (hereinafter “Summers and Smith”).

Prior to inserting the DNA sequence encoding the protein into thebaculovirus genome, the above described components, comprising apromoter, leader (if desired), coding sequence of interest, andtranscription termination sequence, are usually assembled into anintermediate transplacement construct (transfer vector). This constructmay contain a single gene and operably linked regulatory elements;multiple genes, each with its owned set of operably linked regulatoryelements; or multiple genes, regulated by the same set of regulatoryelements. Intermediate transplacement constructs are often maintained ina replicon, such as an extrachromosomal element (eg. plasmids) capableof stable maintenance in a host, such as a bacterium. The replicon willhave a replication system, thus allowing it to be maintained in asuitable host for cloning and amplification.

Currently, the most commonly used transfer vector for introducingforeign genes into AcNPV is pAc373. Many other vectors, known to thoseof skill in the art, have also been designed. These include, forexample, pVL985 (which alters the polyhedrin start codon from ATG toATT, and which introduces a BamHI cloning site 32 basepairs downstreamfrom the ATT; see Luckow and Summers, Virology (1989) 17:31.

The plasmid usually also contains the polyhedrin polyadenylation signal(Miller et al. (1988) Ann. Rev. Microbiol., 42:177) and a prokaryoticampicillin-resistance (amp) gene and origin of replication for selectionand propagation in E. coli.

Baculovirus transfer vectors usually contain a baculovirus promoter. Abaculovirus promoter is any DNA sequence capable of binding abaculovirus RNA polymerase and initiating the downstream (5′ to 3′)transcription of a coding sequence (eg. structural gene) into mRNA. Apromoter will have a transcription initiation region which is usuallyplaced proximal to the 5′ end of the coding sequence. This transcriptioninitiation region usually includes an RNA polymerase binding site and atranscription initiation site. A baculovirus transfer vector may alsohave a second domain called an enhancer, which, if present, is usuallydistal to the structural gene. Expression may be either regulated orconstitutive.

Structural genes, abundantly transcribed at late times in a viralinfection cycle, provide particularly useful promoter sequences.Examples include sequences derived from the gene encoding the viralpolyhedron protein, Friesen et al., (1986) “The Regulation ofBaculovirus Gene Expression,” in: The Molecular Biology of Baculoviruses(ed. Walter Doerfler); EPO Publ. Nos. 127 839 and 155 476; and the geneencoding the p10 protein, Vlak et al., (1988), J. Gen. Virol. 69:765.

DNA encoding suitable signal sequences can be derived from genes forsecreted insect or baculovirus proteins, such as the baculoviruspolyhedrin gene (Carbonell et al. (1988) Gene, 73:409). Alternatively,since the signals for mammalian cell posttranslational modifications(such as signal peptide cleavage, proteolytic cleavage, andphosphorylation) appear to be recognized by insect cells, and thesignals required for secretion and nuclear accumulation also appear tobe conserved between the invertebrate cells and vertebrate cells,leaders of non-insect origin, such as those derived from genes encodinghuman α-interferon, Maeda et al., (1985), Nature 315:592; humangastrin-releasing peptide, Lebacq-Verheyden et al., (1988), Molec. Cell.Biol. 8:3129; human IL-2, Smith et al., (1985) Proc. Nat'l. Acad. Sci.USA, 82:8404; mouse IL-3, (Miyajima et al., (1987) Gene 58:273; andhuman glucocerebrosidase, Martin et al. (1988) DNA, 7:99, can also beused to provide for secretion in insects.

A recombinant polypeptide or polyprotein may be expressedintracellularly or, if it is expressed with the proper regulatorysequences, it can be secreted. Good intracellular expression of nonfusedforeign proteins usually requires heterologous genes that ideally have ashort leader sequence containing suitable translation initiation signalspreceding an ATG start signal. If desired, methionine at the N-terminusmay be cleaved from the mature protein by in vitro incubation withcyanogen bromide.

Alternatively, recombinant polyproteins or proteins which are notnaturally secreted can be secreted from the insect cell by creatingchimeric DNA molecules that encode a fusion protein comprised of aleader sequence fragment that provides for secretion of the foreignprotein in insects. The leader sequence fragment usually encodes asignal peptide comprised of hydrophobic amino acids which direct thetranslocation of the protein into the endoplasmic reticulum.

After insertion of the DNA sequence and/or the gene encoding theexpression product precursor of the protein, an insect cell host isco-transformed with the heterologous DNA of the transfer vector and thegenomic DNA of wild type baculovirus—usually by co-transfection. Thepromoter and transcription termination sequence of the construct willusually comprise a 2-5 kb section of the baculovirus genome. Methods forintroducing heterologous DNA into the desired site in the baculovirusvirus are known in the art. (See Summers and Smith supra; Ju et al.(1987); Smith et al., Mol. Cell. Biol. (1983) 3:2156; and Luckow andSummers (1989)). For example, the insertion can be into a gene such asthe polyhedrin gene, by homologous double crossover recombination;insertion can also be into a restriction enzyme site engineered into thedesired baculovirus gene. Miller et al., (1989), Bioessays 4:91. The DNAsequence, when cloned in place of the polyhedrin gene in the expressionvector, is flanked both 5′ and 3′ by polyhedrin-specific sequences andis positioned downstream of the polyhedrin promoter.

The newly formed baculovirus expression vector is subsequently packagedinto an infectious recombinant baculovirus. Homologous recombinationoccurs at low frequency (between about 1% and about 5%); thus, themajority of the virus produced after cotransfection is still wild-typevirus. Therefore, a method is necessary to identify recombinant viruses.An advantage of the expression system is a visual screen allowingrecombinant viruses to be distinguished. The polyhedrin protein, whichis produced by the native virus, is produced at very high levels in thenuclei of infected cells at late times after viral infection.Accumulated polyhedrin protein forms occlusion bodies that also containembedded particles. These occlusion bodies, up to 15 μm in size, arehighly refractile, giving them a bright shiny appearance that is readilyvisualized under the light microscope. Cells infected with recombinantviruses lack occlusion bodies. To distinguish recombinant virus fromwild-type virus, the transfection supernatant is plagued onto amonolayer of insect cells by techniques known to those skilled in theart. Namely, the plaques are screened under the light microscope for thepresence (indicative of wild-type virus) or absence (indicative ofrecombinant virus) of occlusion bodies. “Current Protocols inMicrobiology” Vol. 2 (Ausubel et al. eds) at 16.8 (Supp. 10, 1990);Summers and Smith, supra; Miller et al. (1989).

Recombinant baculovirus expression vectors have been developed forinfection into several insect cells. For example, recombinantbaculoviruses have been developed for, inter alia: Aedes aegypti,Autographa californica, Bombyx mori, Drosophila melanogaster, Spodopterafrugiperda, and Trichoplusia ni (WO 89/046699; Carbonell et al., (1985)J. Virol. 56:153; Wright (1986) Nature 321:718; Smith et al., (1983)Mol. Cell. Biol. 3:2156; and see generally, Fraser, et al. (1989) InVitro Cell. Dev. Biol. 25:225).

Cells and cell culture media are commercially available for both directand fusion expression of heterologous polypeptides in abaculovirus/expression system; cell culture technology is generallyknown to those skilled in the art. See, eg. Summers and Smith supra.

The modified insect cells may then be grown in an appropriate nutrientmedium, which allows for stable maintenance of the plasmid(s) present inthe modified insect host. Where the expression product gene is underinducible control, the host may be grown to high density, and expressioninduced. Alternatively, where expression is constitutive, the productwill be continuously expressed into the medium and the nutrient mediummust be continuously circulated, while removing the product of interestand augmenting depleted nutrients. The product may be purified by suchtechniques as chromatography, eg. HPLC, affinity chromatography, ionexchange chromatography, etc.; electrophoresis; density gradientcentrifugation; solvent extraction, or the like. As appropriate, theproduct may be further purified, as required, so as to removesubstantially any insect proteins which are also secreted in the mediumor result from lysis of insect cells, so as to provide a product whichis at least substantially free of host debris, eg. proteins, lipids andpolysaccharides.

In order to obtain protein expression, recombinant host cells derivedfrom the transformants are incubated under conditions which allowexpression of the recombinant protein encoding sequence. Theseconditions will vary, dependent upon the host cell selected. However,the conditions are readily ascertainable to those of ordinary skill inthe art, based upon what is known in the art.

iii. Plant Systems

There are many plant cell culture and whole plant genetic expressionsystems known in the art. Exemplary plant cellular genetic expressionsystems include those described in patents, such as: U.S. Pat. No.5,693,506; U.S. Pat. No. 5,659,122; and U.S. Pat. No. 5,608,143.Additional examples of genetic expression in plant cell culture has beendescribed by Zenk, Phytochemistry 30:3861-3863 (1991). Descriptions ofplant protein signal peptides may be found in addition to the referencesdescribed above in Vaulcombe et al., Mol. Gen. Genet. 209:33-40 (1987);Chandler et al., Plant Molecular Biology 3:407-418 (1984); Rogers, J.Biol. Chem. 260:3731-3738 (1985); Rothstein et al., Gene 55:353-356(1987); Whittier et al., Nucleic Acids Research 15:2515-2535 (1987);Wirsel et al., Molecular Microbiology 3:3-14 (1989); Yu et al., Gene122:247-253 (1992). A description of the regulation of plant geneexpression by the phytohormone, gibberellic acid and secreted enzymesinduced by gibberellic acid can be found in R. L. Jones and J.MacMillin, Gibberellins: in: Advanced Plant Physiology, Malcolm B.Wilkins, ed., 1984 Pitman Publishing Limited, London, pp. 21-52.References that describe other metabolically-regulated genes: Sheen,Plant Cell, 2:1027-1038 (1990); Maas et al., EMBO J. 9:3447-3452 (1990);Benkel and Hickey, Proc. Natl. Acad. Sci. 84:1337-1339 (1987)

Typically, using techniques known in the art, a desired polynucleotidesequence is inserted into an expression cassette comprising geneticregulatory elements designed for operation in plants. The expressioncassette is inserted into a desired expression vector with companionsequences upstream and downstream from the expression cassette suitablefor expression in a plant host. The companion sequences will be ofplasmid or viral origin and provide necessary characteristics to thevector to permit the vectors to move DNA from an original cloning host,such as bacteria, to the desired plant host. The basic bacterial/plantvector construct will preferably provide a broad host range prokaryotereplication origin; a prokaryote selectable marker; and, forAgrobacterium transformations, T DNA sequences forAgrobacterium-mediated transfer to plant chromosomes. Where theheterologous gene is not readily amenable to detection, the constructwill preferably also have a selectable marker gene suitable fordetermining if a plant cell has been transformed. A general review ofsuitable markers, for example for the members of the grass family, isfound in Wilmink and Dons, 1993, Plant Mol. Biol. Repir, 11(2):165-185.

Sequences suitable for permitting integration of the heterologoussequence into the plant genome are also recommended. These might includetransposon sequences and the like for homologous recombination as wellas Ti sequences which permit random insertion of a heterologousexpression cassette into a plant genome. Suitable prokaryote selectablemarkers include resistance toward antibiotics such as ampicillin ortetracycline. Other DNA sequences encoding additional functions may alsobe present in the vector, as is known in the art.

The nucleic acid molecules of the subject invention may be included intoan expression cassette for expression of the protein(s) of interest.Usually, there will be only one expression cassette, although two ormore are feasible. The recombinant expression cassette will contain inaddition to the heterologous protein encoding sequence the followingelements, a promoter region, plant 5′ untranslated sequences, initiationcodon depending upon whether or not the structural gene comes equippedwith one, and a transcription and translation termination sequence.Unique restriction enzyme sites at the 5′ and 3′ ends of the cassetteallow for easy insertion into a pre-existing vector.

A heterologous coding sequence may be for any protein relating to thepresent invention. The sequence encoding the protein of interest willencode a signal peptide which allows processing and translocation of theprotein, as appropriate, and will usually lack any sequence which mightresult in the binding of the desired protein of the invention to amembrane. Since, for the most part, the transcriptional initiationregion will be for a gene which is expressed and translocated duringgermination, by employing the signal peptide which provides fortranslocation, one may also provide for translocation of the protein ofinterest. In this way, the protein(s) of interest will be translocatedfrom the cells in which they are expressed and may be efficientlyharvested. Typically secretion in seeds are across the aleurone orscutellar epithelium layer into the endosperm of the seed. While it isnot required that the protein be secreted from the cells in which theprotein is produced, this facilitates the isolation and purification ofthe recombinant protein.

Since the ultimate expression of the desired gene product will be in aeucaryotic cell it is desirable to determine whether any portion of thecloned gene contains sequences which will be processed out as introns bythe host's splicosome machinery. If so, site-directed mutagenesis of the“intron” region may be conducted to prevent losing a portion of thegenetic message as a false intron code, Reed and Maniatis, Cell41:95-105, 1985.

The vector can be microinjected directly into plant cells by use ofmicropipettes to mechanically transfer the recombinant DNA. Crossway,Mol. Gen. Genet, 202:179-185, 1985. The genetic material may also betransferred into the plant cell by using polyethylene glycol, Krens, etal., Nature, 296, 72-74, 1982. Another method of introduction of nucleicacid segments is high velocity ballistic penetration by small particleswith the nucleic acid either within the matrix of small beads orparticles, or on the surface, Klein, et al., Nature, 327, 70-73, 1987and Knudsen and Muller, 1991, Planta, 185:330-336 teaching particlebombardment of barley endosperm to create transgenic barley. Yet anothermethod of introduction would be fusion of protoplasts with otherentities, either minicells, cells, lysosomes or other fusiblelipid-surfaced bodies, Fraley, et al., Proc. Natl. Acad. Sci. USA, 79,1859-1863, 1982.

The vector may also be introduced into the plant cells byelectroporation. (Fromm et al., Proc. Natl. Acad. Sci. USA 82:5824,1985). In this technique, plant protoplasts are electroporated in thepresence of plasmids containing the gene construct. Electrical impulsesof high field strength reversibly permeabilize biomembranes allowing theintroduction of the plasmids. Electroporated plant protoplasts reformthe cell wall, divide, and form plant callus.

All plants from which protoplasts can be isolated and cultured to givewhole regenerated plants can be transformed by the present invention sothat whole plants are recovered which contain the transferred gene. Itis known that practically all plants can be regenerated from culturedcells or tissues, including but not limited to all major species ofsugarcane, sugar beet, cotton, fruit and other trees, legumes andvegetables. Some suitable plants include, for example, species from thegenera Fragaria, Lotus, Medicago, Onobrychis, Trifolium, Trigonella,Vigna, Citrus, Linum, Geranium, Manihot, Daucus, Arabidopsis, Brassica,Raphanus, Sinapis, Atropa, Capsicum, Datura, Hyoscyamus, Lycopersion,Nicotiana, Solanum, Petunia, Digitalis, Majorana, Cichorium, Helianthus,Lactuca, Bromus, Asparagus, Antirrhinum, Hererocallis, Nemesia,Pelargonium, Panicum, Pennisetum, Ranunculus, Senecio, Salpiglossis,Cucumis, Browaalia, Glycine, Lolium, Zea, Triticum, Sorghum, and Datura.

Means for regeneration vary from species to species of plants, butgenerally a suspension of transformed protoplasts containing copies ofthe heterologous gene is first provided. Callus tissue is formed andshoots may be induced from callus and subsequently rooted.Alternatively, embryo formation can be induced from the protoplastsuspension. These embryos germinate as natural embryos to form plants.The culture media will generally contain various amino acids andhormones, such as auxin and cytokinins. It is also advantageous to addglutamic acid and proline to the medium, especially for such species ascorn and alfalfa. Shoots and roots normally develop simultaneously.Efficient regeneration will depend on the medium, on the genotype, andon the history of the culture. If these three variables are controlled,then regeneration is fully reproducible and repeatable.

In some plant cell culture systems, the desired protein of the inventionmay be excreted or alternatively, the protein may be extracted from thewhole plant. Where the desired protein of the invention is secreted intothe medium, it may be collected. Alternatively, the embryos andembryoless-half seeds or other plant tissue may be mechanicallydisrupted to release any secreted protein between cells and tissues. Themixture may be suspended in a buffer solution to retrieve solubleproteins. Conventional protein isolation and purification methods willbe then used to purify the recombinant protein. Parameters of time,temperature pH, oxygen, and volumes will be adjusted through routinemethods to optimize expression and recovery of heterologous protein.

iv. Bacterial Systems

Bacterial expression techniques are known in the art. A bacterialpromoter is any DNA sequence capable of binding bacterial RNA polymeraseand initiating the downstream (3′) transcription of a coding sequence(eg. structural gene) into mRNA. A promoter will have a transcriptioninitiation region which is usually placed proximal to the 5′ end of thecoding sequence. This transcription initiation region usually includesan RNA polymerase binding site and a transcription initiation site. Abacterial promoter may also have a second domain called an operator,that may overlap an adjacent RNA polymerase binding site at which RNAsynthesis begins. The operator permits negative regulated (inducible)transcription, as a gene repressor protein may bind the operator andthereby inhibit transcription of a specific gene. Constitutiveexpression may occur in the absence of negative regulatory elements,such as the operator. In addition, positive regulation may be achievedby a gene activator protein binding sequence, which, if present isusually proximal (5′) to the RNA polymerase binding sequence. An exampleof a gene activator protein is the catabolite activator protein (CAP),which helps initiate transcription of the lac operon in Escherichia coli(E. coli) [Raibaud et al. (1984) Annu. Rev. Genet. 18:173]. Regulatedexpression may therefore be either positive or negative, thereby eitherenhancing or reducing transcription.

Sequences encoding metabolic pathway enzymes provide particularly usefulpromoter sequences. Examples include promoter sequences derived fromsugar metabolizing enzymes, such as galactose, lactose (lac) [Chang etal. (1977) Nature 198:1056], and maltose. Additional examples includepromoter sequences derived from biosynthetic enzymes such as tryptophan(trp) [Goeddel et al. (1980) Nuc. Acids Res. 8:4057; Yelverton et al.(1981) Nucl. Acids Res. 9:731; U.S. Pat. No. 4,738,921; EP-A-0036776 andEP-A-0121775]. The g-laotamase (bla) promoter system [Weissmann (1981)“The cloning of interferon and other mistakes.” In Interferon 3 (ed. I.Gresser)], bacteriophage lambda PL [Shimatake et al. (1981) Nature292:128] and T5 [U.S. Pat. No. 4,689,406] promoter systems also provideuseful promoter sequences.

In addition, synthetic promoters which do not occur in nature alsofunction as bacterial promoters. For example, transcription activationsequences of one bacterial or bacteriophage promoter may be joined withthe operon sequences of another bacterial or bacteriophage promoter,creating a synthetic hybrid promoter [U.S. Pat. No. 4,551,433]. Forexample, the tac promoter is a hybrid trp-lac promoter comprised of bothtrp promoter and lac operon sequences that is regulated by the lacrepressor [Amann et al. (1983) Gene 25:167; de Boer et al. (1983) Proc.Natl. Acad. Sci. 80:21]. Furthermore, a bacterial promoter can includenaturally occurring promoters of non-bacterial origin that have theability to bind bacterial RNA polymerase and initiate transcription. Anaturally occurring promoter of non-bacterial origin can also be coupledwith a compatible RNA polymerase to produce high levels of expression ofsome genes in prokaryotes. The bacteriophage T7 RNA polymerase/promotersystem is an example of a coupled promoter system [Studier et al. (1986)J. Mol. Biol. 189:113; Tabor et al. (1985) Proc Natl. Acad. Sci.82:1074]. In addition, a hybrid promoter can also be comprised of abacteriophage promoter and an E. coli operator region (EPO-A-0 267 851).

In addition to a functioning promoter sequence, an efficient ribosomebinding site is also useful for the expression of foreign genes inprokaryotes. In E. coli, the ribosome binding site is called theShine-Dalgarno (SD) sequence and includes an initiation codon (ATG) anda sequence 3-9 nucleotides in length located 3-11 nucleotides upstreamof the initiation codon [Shine et al. (1975) Nature 254:34]. The SDsequence is thought to promote binding of mRNA to the ribosome by thepairing of bases between the SD sequence and the 3′ and of E. coli 16SrRNA [Steitz et al. (1979) “Genetic signals and nucleotide sequences inmessenger RNA.” In Biological Regulation and Development: GeneExpression (ed. R. F. Goldberger)]. To express eukaryotic genes andprokaryotic genes with weak ribosome-binding site [Sambrook et al.(1989) “Expression of cloned genes in Escherichia coli.” In MolecularCloning: A Laboratory Manual].

A DNA molecule may be expressed intracellularly. A promoter sequence maybe directly linked with the DNA molecule, in which case the first aminoacid at the N-terminus will always be a methionine, which is encoded bythe ATG start codon. If desired, methionine at the N-terminus may becleaved from the protein by in vitro incubation with cyanogen bromide orby either in vivo on in vitro incubation with a bacterial methionineN-terminal peptidase (EPO-A-0 219 237).

Fusion proteins provide an alternative to direct expression. Usually, aDNA sequence encoding the N-terminal portion of an endogenous bacterialprotein, or other stable protein, is fused to the 5′ end of heterologouscoding sequences. Upon expression, this construct will provide a fusionof the two amino acid sequences. For example, the bacteriophage lambdacell gene can be linked at the 5′ terminus of a foreign gene andexpressed in bacteria. The resulting fusion protein preferably retains asite for a processing enzyme (factor Xa) to cleave the bacteriophageprotein from the foreign gene [Nagai et al. (1984) Nature 309:810].Fusion proteins can also be made with sequences from the lacZ [Jia etal. (1987) Gene 60:197], trpE [Allen et al. (1987) J. Biotechnol. 5:93;Makoff et al. (1989) J. Gen. Microbiol. 135:11], and Chey [EP-A-0 324647] genes. The DNA sequence at the junction of the two amino acidsequences may or may not encode a cleavable site. Another example is aubiquitin fusion protein. Such a fusion protein is made with theubiquitin region that preferably retains a site for a processing enzyme(eg. ubiquitin specific processing-protease) to cleave the ubiquitinfrom the foreign protein. Through this method, native foreign proteincan be isolated [Miller et al. (1989) Bio/Technology 7:698].

Alternatively, foreign proteins can also be secreted from the cell bycreating chimeric DNA molecules that encode a fusion protein comprisedof a signal peptide sequence fragment that provides for secretion of theforeign protein in bacteria [U.S. Pat. No. 4,336,336]. The signalsequence fragment usually encodes a signal peptide comprised ofhydrophobic amino acids which direct the secretion of the protein fromthe cell. The protein is either secreted into the growth media(gram-positive bacteria) or into the periplasmic space, located betweenthe inner and outer membrane of the cell (gram-negative bacteria).Preferably there are processing sites, which can be cleaved either invivo or in vitro encoded between the signal peptide fragment and theforeign gene.

DNA encoding suitable signal sequences can be derived from genes forsecreted bacterial proteins, such as the E. coli outer membrane proteingene (ompA) [Masui et al. (1983), in: Experimental Manipulation of GeneExpression; Ghrayeb et al. (1984) EMBO J. 3:2437] and the E. colialkaline phosphatase signal sequence (phoA) [Oka et al. (1985) Proc.Natl. Acad. Sci. 82:7212]. As an additional example, the signal sequenceof the alpha-amylase gene from various Bacillus strains can be used tosecrete heterologous proteins from B. subtilis [Palva et al. (1982)Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 244 042].

Usually, transcription termination sequences recognized by bacteria areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Transcription termination sequencesfrequently include DNA sequences of about 50 nucleotides capable offorming stem loop structures that aid in terminating transcription.Examples include transcription termination sequences derived from geneswith strong promoters, such as the trp gene in E. coli as well as otherbiosynthetic genes.

Usually, the above described components, comprising a promoter, signalsequence (if desired), coding sequence of interest, and transcriptiontermination sequence, are put together into expression constructs.Expression constructs are often maintained in a replicon, such as anextrachromosomal element (eg. plasmids) capable of stable maintenance ina host, such as bacteria. The replicon will have a replication system,thus allowing it to be maintained in a prokaryotic host either forexpression or for cloning and amplification. In addition, a replicon maybe either a high or low copy number plasmid. A high copy number plasmidwill generally have a copy number ranging from about 5 to about 200, andusually about 10 to about 150. A host containing a high copy numberplasmid will preferably contain at least about 10, and more preferablyat least about 20 plasmids. Either a high or low copy number vector maybe selected, depending upon the effect of the vector and the foreignprotein on the host.

Alternatively, the expression constructs can be integrated into thebacterial genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to the bacterial chromosomethat allows the vector to integrate. Integrations appear to result fromrecombinations between homologous DNA in the vector and the bacterialchromosome. For example, integrating vectors constructed with DNA fromvarious Bacillus strains integrate into the Bacillus chromosome (EP-A-0127 328). Integrating vectors may also be comprised of bacteriophage ortransposon sequences.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of bacterialstrains that have been transformed. Selectable markers can be expressedin the bacterial host and may include genes which render bacteriaresistant to drugs such as ampicillin, chloramphenicol, erythromycin,kanamycin (neomycin), and tetracycline [Davies et al. (1978) Annu. Rev.Microbiol. 32:469]. Selectable markers may also include biosyntheticgenes, such as those in the histidine, tryptophan, and leucinebiosynthetic pathways.

Alternatively, some of the above described components can be puttogether in transformation vectors. Transformation vectors are usuallycomprised of a selectable market that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extra-chromosomalreplicons or integrating vectors, have been developed for transformationinto many bacteria. For example, expression vectors have been developedfor, inter alia, the following bacteria: Bacillus subtilis [Palva et al.(1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259 and EP-A-0 063953; WO 84/04541], Escherichia coli [Shimatake et al. (1981) Nature292:128; Amann et al. (1985) Gene 40:183; Studier et al. (1986) J. Mol.Biol. 189:113; EP-A-0 036 776, EP-A-0 136 829 and EP-A-0 136 907],Streptococcus cremoris [Powell et al. (1988) Appl. Environ. Microbiol.54:655]; Streptococcus lividans [Powell et al. (1988) Appl. Environ.Microbiol. 54:655], Streptomyces lividans [U.S. Pat. No. 4,745,056].

Methods of introducing exogenous DNA into bacterial hosts are well-knownin the art, and usually include either the transformation of bacteriatreated with CaCl₂ or other agents, such as divalent cations and DMSO.DNA can also be introduced into bacterial cells by electroporation.Transformation procedures usually vary with the bacterial species to betransformed. See eg. [Masson et al. (1989) FEMS Microbiol. Lett. 60:273;Palva et al. (1982) Proc. Natl. Acad. Sci. USA 79:5582; EP-A-0 036 259and EP-A-0 063 953; WO 84/04541, Bacillus], [Miller et al. (1988) Proc.Natl. Acad. Sci. 85:856; Wang et al. (1990) J. Bacteriol. 172:949,Campylobacter], [Cohen et al. (1973) Proc. Natl. Acad. Sci. 69:2110;Dower et al. (1988) Nucleic Acids Res. 16:6127; Kushner (1978) “Animproved method for transformation of Escherichia coli withColE1-derived plasmids. In Genetic Engineering: Proceedings of theInternational Symposium on Genetic Engineering (eds. H. W. Boyer and S.Nicosia); Mandel et al. (1970) J. Mol. Biol. 53:159; Taketo (1988)Biochim. Biophys. Acta 949:318; Escherichia], [Chassy et al. (1987) FEMSMicrobiol. Lett. 44:173 Lactobacillus]; [Fiedler et al. (1988) Anal.Biochem 170:38, Pseudomonas]; [Augustin et al. (1990) FEMS Microbiol.Lett. 66:203, Staphylococcus], [Barany et al. (1980) J. Bacteriol.144:698; Harlander (1987) “Transformation of Streptococcus lactis byelectroporation, in: Streptococcal Genetics (ed. J. Ferretti and R.Curtiss III); Perry et al. (1981) Infect. Immun. 32:1295; Powell et al.(1988) Appl. Environ. Microbiol. 54:655; Somkuti et al. (1987) Proc. 4thEvr. Cong. Biotechnology 1:412, Streptococcus].

v. Yeast Expression

Yeast expression systems are also known to one of ordinary skill in theart. A yeast promoter is any DNA sequence capable of binding yeast RNApolymerase and initiating the downstream (3′) transcription of a codingsequence (eg. structural gene) into mRNA. A promoter will have atranscription initiation region which is usually placed proximal to the5′ end of the coding sequence. This transcription initiation regionusually includes an RNA polymerase binding site (the “TATA Box”) and atranscription initiation site. A yeast promoter may also have a seconddomain called an upstream activator sequence (UAS), which, if present,is usually distal to the structural gene. The UAS permits regulated(inducible) expression. Constitutive expression occurs in the absence ofa UAS. Regulated expression may be either positive or negative, therebyeither enhancing or reducing transcription.

Yeast is a fermenting organism with an active metabolic pathway,therefore sequences encoding enzymes in the metabolic pathway provideparticularly useful promoter sequences. Examples include alcoholdehydrogenase (ADH) (EP-A-6 284 044), enolase, glucokinase,glucose-6-phosphate isomerase, glyceraldehyde-3-phosphate-dehydrogenase(GAP or GAPDH), hexokinase, phosphofructokinase, 3-phosphoglyceratemutase, and pyruvate kinase (PyK) (EPO-A-0 329 203). The yeast PHO5gene, encoding acid phosphatase, also provides useful promoter sequences[Myanohara et al. (1983) Proc. Natl. Acad. Sci. USA 80:1].

In addition, synthetic promoters which do not occur in nature alsofunction as yeast promoters. For example, UAS sequences of one yeastpromoter may be joined with the transcription activation region ofanother yeast promoter, creating a synthetic hybrid promoter. Examplesof such hybrid promoters include the ADH regulatory sequence linked tothe GAP transcription activation region (U.S. Pat. Nos. 4,876,197 and4,880,734). Other examples of hybrid promoters include promoters whichconsist of the regulatory sequences of either the ADH2, GAL4, GAL10, ORPHO5 genes, combined with the transcriptional activation region of aglycolytic enzyme gene such as GAP or PyK (EP-A-0 164 556). Furthermore,a yeast promoter can include naturally occurring promoters of non-yeastorigin that have the ability to bind yeast RNA polymerase and initiatetranscription. Examples of such promoters include, inter alia, [Cohen etal. (1980) Proc. Natl. Acad. Sci. USA 77:1078; Henikoff et al. (1981)Nature 283:835; Hollenberg et al. (1981) Curr. Topics Microbiol.Immunol. 96:119; Hollenberg et al. (1979) “The Expression of BacterialAntibiotic Resistance Genes in the Yeast Saccharomyces cerevisiae,” in:Plasmids of Medical, Environmental and Commercial Importance (eds. K. N.Timmis and A. Puhler); Mercerau-Puigalon et al. (1980) Gene 11:163;Panthier et al. (1980) Curr. Genet. 2:109;].

A DNA molecule may be expressed intracellularly in yeast. A promotersequence may be directly linked with the DNA molecule, in which case thefirst amino acid at the N-terminus of the recombinant protein willalways be a methionine, which is encoded by the ATG start codon. Ifdesired, methionine at the N-terminus may be cleaved from the protein byin vitro incubation with cyanogen bromide.

Fusion proteins provide an alternative for yeast expression systems, aswell as in mammalian, baculovirus, and bacterial expression systems.Usually, a DNA sequence encoding the N-terminal portion of an endogenousyeast protein, or other stable protein, is fused to the 5′ end ofheterologous coding sequences. Upon expression, this construct willprovide a fusion of the two amino acid sequences. For example, the yeastor human superoxide dismutase (SOD) gene, can be linked at the 5′terminus of a foreign gene and expressed in yeast. The DNA sequence atthe junction of the two amino acid sequences may or may not encode acleavable site. See eg. EP-A-0 196 056. Another example is a ubiquitinfusion protein. Such a fusion protein is made with the ubiquitin regionthat preferably retains a site for a processing enzyme (eg.ubiquitin-specific processing protease) to cleave the ubiquitin from theforeign protein. Through this method, therefore, native foreign proteincan be isolated (eg. WO88/024066).

Alternatively, foreign proteins can also be secreted from the cell intothe growth media by creating chimeric DNA molecules that encode a fusionprotein comprised of a leader sequence fragment that provide forsecretion in yeast of the foreign protein. Preferably, there areprocessing sites encoded between the leader fragment and the foreigngene that can be cleaved either in vivo or in vitro. The leader sequencefragment usually encodes a signal peptide comprised of hydrophobic aminoacids which direct the secretion of the protein from the cell.

DNA encoding suitable signal sequences can be derived from genes forsecreted yeast proteins, such as the yeast invertase gene (EP-A-0 012873; JPO. 62,096,086) and the A-factor gene (U.S. Pat. No. 4,588,684).Alternatively, leaders of non-yeast origin, such as an interferonleader, exist that also provide for secretion in yeast (EP-A-0 060 057).

A preferred class of secretion leaders are those that employ a fragmentof the yeast alpha-factor gene, which contains both a “pre” signalsequence, and a “pro” region. The types of alpha-factor fragments thatcan be employed include the full-length pre-pro alpha factor leader(about 83 amino acid residues) as well as truncated alpha-factor leaders(usually about 25 to about 50 amino acid residues) (U.S. Pat. Nos.4,546,083 and 4,870,008; EP-A-0 324 274). Additional leaders employingan alpha-factor leader fragment that provides for secretion includehybrid alpha-factor leaders made with a presequence of a first yeast,but a pro-region from a second yeast alphafactor. (eg. see WO 89/02463.)

Usually, transcription termination sequences recognized by yeast areregulatory regions located 3′ to the translation stop codon, and thustogether with the promoter flank the coding sequence. These sequencesdirect the transcription of an mRNA which can be translated into thepolypeptide encoded by the DNA. Examples of transcription terminatorsequence and other yeast-recognized termination sequences, such as thosecoding for glycolytic enzymes.

Usually, the above described components, comprising a promoter, leader(if desired), coding sequence of interest, and transcription terminationsequence, are put together into expression constructs. Expressionconstructs are often maintained in a replicon, such as anextrachromosomal element (eg. plasmids) capable of stable maintenance ina host, such as yeast or bacteria. The replicon may have two replicationsystems, thus allowing it to be maintained, for example, in yeast forexpression and in a prokaryotic host for cloning and amplification.Examples of such yeast-bacteria shuttle vectors include YEp24 [Botsteinet al. (1979) Gene 8:17-24], pCl/1 [Brake et al. (1984) Proc. Natl.Acad. Sci USA 81:4642-4646], and YRp17 [Stinchcomb et al. (1982) J. Mol.Biol. 158:157]. In addition, a replicon may be either a high or low copynumber plasmid. A high copy number plasmid will generally have a copynumber ranging from about 5 to about 200, and usually about 10 to about150. A host containing a high copy number plasmid will preferably haveat least about 10, and more preferably at least about 20. Enter a highor low copy number vector may be selected, depending upon the effect ofthe vector and the foreign protein on the host. See eg. Brake et al.,supra.

Alternatively, the expression constructs can be integrated, into theyeast genome with an integrating vector. Integrating vectors usuallycontain at least one sequence homologous to a yeast chromosome thatallows the vector to integrate, and preferably contain two homologoussequences flanking the expression construct. Integrations appear toresult from recombinations between homologous DNA in the vector and theyeast chromosome [Orr-Weaver et al. (1983) Methods in Enzymol.101:228-245]. An integrating vector may be directed to a specific locusin yeast by selecting the appropriate homologous sequence for inclusionin the vector. See Orr-Weaver et al., supra. One or more expressionconstruct may integrate, possibly affecting levels of recombinantprotein produced [Rine et al. (1983) Proc. Natl. Acad. Sci. USA80:6750]. The chromosomal sequences included in the vector can occureither as a single segment in the vector, which results in theintegration of the entire vector, or two segments homologous to adjacentsegments in the chromosome and flanking the expression construct in thevector, which can result in the stable integration of only theexpression construct.

Usually, extrachromosomal and integrating expression constructs maycontain selectable markers to allow for the selection of yeast strainsthat have been transformed. Selectable markers may include biosyntheticgenes that can be expressed in the yeast host, such as ADE2, HIS4, LEU2,TRP1, and ALG7, and the G418 resistance gene, which confer resistance inyeast cells to tunicamycin and G418, respectively. In addition, asuitable selectable marker may also provide yeast with the ability togrow in the presence of toxic compounds, such as metal. For example, thepresence of CUP1 allows yeast to grow in the presence of copper ions[Butt et al. (1987) Microbiol, Rev. 51:351].

Alternatively, some of the above described components can be puttogether into transformation vectors. Transformation vectors are usuallycomprised of a selectable marker that is either maintained in a repliconor developed into an integrating vector, as described above.

Expression and transformation vectors, either extrachromosomal repliconsor integrating vectors, have been developed for transformation into manyyeasts. For example, expression vectors have been developed for, interalia, the following yeasts: Candida albicans [Kurtz, et al. (1986) Mol.Cell. Biol. 6:142], Candida maltosa [Kunze, et al. (1985) J. BasicMicrobiol. 25:141]. Hansenula polymorpha [Gleeson, et al. (1986) J. Gen.Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302],Kluyveromyces fragilis [Das, et al. (1984) J. Bacteriol. 158:1165],Kluyveromyces lactis [De Louvencourt et al. (1983) J. Bacteriol.154:737; Van den Berg et al. (1990) Bio/Technology 8:135], Pichiaguillerimondii [Kunze et al. (1985) J. Basic Microbiol. 25:141], Pichiapastoris [Cregg, et al. (1985) Mol. Cell. Biol. 5:3376; U.S. Pat. Nos.4,837,148 and 4,929,555], Saccharomyces cerevisiae [Hinnen et al. (1978)Proc. Natl. Acad. Sci. USA 75:1929; Ito et al. (1983) J. Bacteriol.153:163], Schizosaccharomyces pombe [Beach and Nurse (1981) Nature300:706], and Yarrowia lipolytica [Davidow, et al. (1985) Curr. Genet.10:380471 Gaillardin, et al. (1985) Curr. Genet. 10:49].

Methods of introducing exogenous DNA into yeast hosts are well-known inthe art, and usually include either the transformation of spheroplastsor of intact yeast cells treated with alkali cations. Transformationprocedures usually vary with the yeast species to be transformed. Seeeg. [Kurtz et al. (1986) Mol. Cell. Biol. 6:142; Kunze et al. (1985) J.Basic Microbiol. 25:141; Candida]; [Gleeson et al. (1986) J. Gen.Microbiol. 132:3459; Roggenkamp et al. (1986) Mol. Gen. Genet. 202:302;Hansenula]; [Das et al. (1984) J. Bacteriol. 158:1165; De Louvencourt etal. (1983) J. Bacteriol. 154:1165; Van den Berg et al. (1990)Bio/Technology 8:135; Kluyveromyces]; [Cregg et al. (1985) Mol. Cell.Biol. 5-3376; Kunze et al. (1985) J. Basic Microbiol. 25:141; U.S. Pat.Nos. 4,837,148 and 4,929,555; Pichia]; [Hinnen et al. (1978) Proc. Natl.Acad. Sci. USA 75; 1929; Ito et al. (1983) J. Bacteriol. 153:163Saccharomyces]; [Beach and Nurse (1981) Nature 300:706;Schizosaccharomyces]; [Davidow et al. (1985) Curr. Genet. 10:39;Gaillardin et al. (1985) Curr. Genet. 10:49; Yarrowia].

Antibodies

As used herein, the term “antibody” refers to a polypeptide or group ofpolypeptides composed of at least one antibody combining site. An“antibody combining site” is the three-dimensional binding space with aninternal surface shape and charge distribution complementary to thefeatures of an epitope of an antigen, which allows a binding of theantibody with the antigen. “Antibody” includes, for example, vertebrateantibodies, hybrid antibodies, chimeric antibodies, humanisedantibodies, altered antibodies, univalent antibodies, Fab proteins, andsingle domain antibodies.

Antibodies against the proteins of the invention are useful for affinitychromatography, immunoassays, and distinguishing/identifying Neisserialproteins.

Antibodies to the proteins of the invention, both polyclonal andmonoclonal, may be prepared by conventional methods. In general, theprotein is first used to immunize a suitable animal, preferably a mouse,rat, rabbit or goat. Rabbits and goats are preferred for the preparationof polyclonal sera due to the volume of serum obtainable, and theavailability of labeled anti-rabbit and anti-goat antibodies.Immunization is generally performed by mixing or emulsifying the proteinin saline, preferably in an adjuvant such as Freund's complete adjuvant,and injecting the mixture or emulsion parenterally (generallysubcutaneously or intramuscularly). A dose of 50-200·μg/injection istypically sufficient. Immunization is generally boosted 2-6 weeks laterwith one or more injections of the protein in saline, preferably usingFreund's incomplete adjuvant. One may alternatively generate antibodiesby in vitro immunization using methods known in the art, which for thepurposes of this invention is considered equivalent to in vivoimmunization. Polyclonal antisera is obtained by bleeding the immunizedanimal into a glass or plastic container, incubating the blood at 25° C.for one hour, followed by incubating at 4° C. for 2-18 hours. The serumis recovered by centrifugation (eg. 1,000 g for 10 minutes). About 20-50ml per bleed may be obtained from rabbits.

Monoclonal antibodies are prepared using the standard method of Kohler &Milstein [Nature (1975) 256:495-96], or a modification thereof.Typically, a mouse or rat is immunized as described above. However,rather than bleeding the animal to extract serum, the spleen (andoptionally several large lymph nodes) is removed and dissociated intosingle cells. If desired, the spleen cells may be screened (afterremoval of nonspecifically adherent cells) by applying a cell suspensionto a plate or well coated with the protein antigen. B-cells expressingmembrane-bound immunoglobulin specific for the antigen bind to theplate, and are not rinsed away with the rest of the suspension.Resulting B-cells, or all dissociated spleen cells, are then induced tofuse with myeloma cells to form hybridomas, and are cultured in aselective medium (eg. hypoxanthine, aminopterin, thymidine medium,“HAT”). The resulting hybridomas are plated by limiting dilution, andare assayed for the production of antibodies which bind specifically tothe immunizing antigen (and which do not bind to unrelated antigens).The selected MAb-secreting hybridomas are then cultured either in vitro(eg. in tissue culture bottles or hollow fiber reactors), or in vivo (asascites in mice).

If desired, the antibodies (whether polyclonal or monoclonal) may belabeled using conventional techniques. Suitable labels includefluorophores, chromophores, radioactive atoms (particularly ³²P and¹²⁵I), electron-dense reagents, enzymes, and ligands having specificbinding partners. Enzymes are typically detected by their activity. Forexample, horseradish peroxidase is usually detected by its ability toconvert 3,3′,5,5′-tetramethylbenzidine (TMB) to a blue pigment,quantifiable with a spectrophotometer. “Specific binding partner” refersto a protein capable of binding a ligand molecule with high specificity,as for example in the case of an antigen and a monoclonal antibodyspecific therefor. Other specific binding partners include biotin andavidin or streptavidin, IgG and protein A, and the numerousreceptor-ligand couples known in the art. It should be understood thatthe above description is not meant to categorize the various labels intodistinct classes, as the same label may serve in several differentmodes. For example, ¹²⁵I may serve as a radioactive label or as anelectron-dense reagent. HRP may serve as enzyme or as antigen for a MAb.Further, one may combine various labels for desired effect. For example,MAbs and avidin also require labels in the practice of this invention:thus, one might label a MAb with biotin, and detect its presence withavidin labeled with ¹²⁵I, or with an anti-biotin MAb labeled with HRP.Other permutations and possibilities will be readily apparent to thoseof ordinary skill in the art, and are considered as equivalents withinthe scope of the instant invention.

Pharmaceutical Compositions

Pharmaceutical compositions can comprise either polypeptides,antibodies, or nucleic acid of the invention. The pharmaceuticalcompositions will comprise a therapeutically effective amount of eitherpolypeptides, antibodies, or polynucleotides of the claimed invention.

The term “therapeutically effective amount” as used herein refers to anamount of a therapeutic agent to treat, ameliorate, or prevent a desireddisease or condition, or to exhibit a detectable therapeutic orpreventative effect. The effect can be detected by, for example,chemical markers or antigen levels. Therapeutic effects also includereduction in physical symptoms, such as decreased body temperature. Theprecise effective amount for a subject will depend upon the subject'ssize and health, the nature and extent of the condition, and thetherapeutics or combination of therapeutics selected for administration.Thus, it is not useful to specify an exact effective amount in advance.However, the effective amount for a given situation can be determined byroutine experimentation and is within the judgement of the clinician.

For purposes of the present invention, an effective dose will be fromabout 0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNAconstructs in the individual to which it is administered.

A pharmaceutical composition can also contain a pharmaceuticallyacceptable carrier. The term “pharmaceutically acceptable carrier”refers to a carrier for administration of a therapeutic agent, such asantibodies or a polypeptide, genes, and other therapeutic agents. Theterm refers to any pharmaceutical carrier that does not itself inducethe production of antibodies harmful to the individual receiving thecomposition, and which may be administered without undue toxicity.Suitable carriers may be large, slowly metabolized macromolecules suchas proteins, polysaccharides, polylactic acids, polyglycolic acids,polymeric amino acids, amino acid copolymers, and inactive virusparticles. Such carriers are well known to those of ordinary skill inthe art.

Pharmaceutically acceptable salts can be used therein, for example,mineral acid salts such as hydrochlorides, hydrobromides, phosphates,sulfates, and the like; and the salts of organic acids such as acetates,propionates, malonates, benzoates, and the like. A thorough discussionof pharmaceutically acceptable excipients is available in Remington'sPharmaceutical Sciences (Mack Pub. Co., N.J. 1991).

Pharmaceutically acceptable carriers in therapeutic compositions maycontain liquids such as water, saline, glycerol and ethanol.Additionally, auxiliary substances, such as wetting or emulsifyingagents, pH buffering substances, and the like, may be present in suchvehicles. Typically, the therapeutic compositions are prepared asinjectables, either as liquid solutions or suspensions; solid formssuitable for solution in, or suspension in, liquid vehicles prior toinjection may also be prepared. Liposomes are included within thedefinition of a pharmaceutically acceptable carrier.

Delivery Methods

Once formulated, the compositions of the invention can be administereddirectly to the subject. The subjects to be treated can be animals; inparticular, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished byinjection; either subcutaneously, intraperitoneally, intravenously orintramuscularly or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Other modes ofadministration include oral and pulmonary administration, suppositories,and transdermal or transcutaneous applications (eg. see WO98/20734),needles, and gene guns or hyposprays. Dosage treatment may be a singledose schedule or a multiple dose schedule.

Vaccines

Vaccines according to the invention may either be prophylactic (ie. toprevent infection) or therapeutic (ie. to treat disease afterinfection).

Such vaccines comprise immunising antigen(s), immunogen(s),polypeptide(s), protein(s) or nucleic acid, usually in combination with“pharmaceutically acceptable carriers,” which include any carrier thatdoes not itself induce the production of antibodies harmful to theindividual receiving the composition. Suitable carriers are typicallylarge, slowly metabolized macromolecules such as proteins,polysaccharides, polylactic acids, polyglycolic acids, polymeric aminoacids, amino acid copolymers, lipid aggregates (such as oil droplets orliposomes), and inactive virus particles. Such carriers are well knownto those of ordinary skill in the art. Additionally, these carriers mayfunction as immunostimulating agents (“adjuvants”). Furthermore, theantigen or immunogen may be conjugated to a bacterial toxoid, such as atoxoid from diphtheria, tetanus, cholera, H. pylori, etc. pathogens.

Preferred adjuvants to enhance effectiveness of the composition include,but are not limited to: (1) aluminum salts (alum), such as aluminumhydroxide, aluminum phosphate, aluminum sulfate, etc; (2) oil-in-wateremulsion formulations (with or without other specific immunostimulatingagents such as muramyl peptides (see below) or bacterial cell wallcomponents), such as for example (a) MF59™ (WO 90/14837; Chapter 10 inVaccine design: the subunit and adjuvant approach, eds. Powell & Newman,Plenum Press 1995), containing 5% Squalene, 0.5% Tween 80, and 0.5% Span85 (optionally containing various amounts of MTP-PE (see below),although not required) formulated into submicron particles using amicrofluidizer such as Model 110Y microfluidizer (Microfluidics, Newton,Mass.), (b) SAF, containing 10% Squalane, 0.4% Tween 80, 5%pluronic-blocked polymer L121, and thr-MDP (see below) eithermicrofluidized into a submicron emulsion or vortexed to generate alarger particle size emulsion, and (c) Ribi™ adjuvant system (RAS),(Ribi Immunochem, Hamilton, Mont.) containing 2% Squalene, 0.2% Tween80, and one or more bacterial cell wall components from the groupconsisting of monophosphorylipid A (MPL), trehalose dimycolate (TDM),and cell wall skeleton (CWS), preferably MPL+CWS (Detox™); (3) saponinadjuvants, such as Stimulon™ (Cambridge Bioscience, Worcester, Mass.)may be used or particles generated therefrom such as ISCOMs(immunostimulating complexes); (4) Complete Freund's Adjuvant (CFA) andIncomplete Freund's Adjuvant (IFA); (5) cytokines, such as interleukins(eg. IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, etc.), interferons (eg.gamma interferon), macrophage colony stimulating factor (M-CSF), tumornecrosis factor (TNF), etc; and (6) other substances that act asimmunostimulating agents to enhance the effectiveness of thecomposition. Alum and MF59™ are preferred.

As mentioned above, muramyl peptides include, but are not limited to,N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP),N-acetyl-normuramyl-L-alanyl-D-isoglutamine (nor-MDP),N-acetylmuramyl-L-alanyl-D-isoglutaminyl-L-alanine-2-(1′-2′-dipalmitoyl-sn-glycero-3-hydroxyphosphoryloxy)-ethylamine(MTP-PE), etc.

The immunogenic compositions (eg. the immunisingantigen/immunogen/polypeptide/protein/nucleic acid, pharmaceuticallyacceptable carrier, and adjuvant) typically will contain diluents, suchas water, saline, glycerol, ethanol, etc. Additionally, auxiliarysubstances, such as wetting or emulsifying agents, pH bufferingsubstances, and the like, may be present in such vehicles.

Typically, the immunogenic compositions are prepared as injectables,either as liquid solutions or suspensions; solid forms suitable forsolution in, or suspension in, liquid vehicles prior to injection mayalso be prepared. The preparation also may be emulsified or encapsulatedin liposomes for enhanced adjuvant effect, as discussed above underpharmaceutically acceptable carriers.

Immunogenic compositions used as vaccines comprise an immunologicallyeffective amount of the antigenic or immunogenic polypeptides, as wellas any other of the above-mentioned components, as needed. By“immunologically effective amount”, it is meant that the administrationof that amount to an individual, either in a single dose or as part of aseries, is effective for treatment or prevention. This amount variesdepending upon the health and physical condition of the individual to betreated, the taxonomic group of individual to be treated (eg. nonhumanprimate, primate, etc.), the capacity of the individual's immune systemto synthesize antibodies, the degree of protection desired, theformulation of the vaccine, the treating doctor's assessment of themedical situation, and other relevant factors. It is expected that theamount will fall in a relatively broad range that can be determinedthrough routine trials.

The immunogenic compositions are conventionally administeredparenterally, eg. by injection, either subcutaneously, intramuscularly,or transdermally/transcutaneously (eg. WO98/20734). Additionalformulations suitable for other modes of administration include oral andpulmonary formulations, suppositories, and transdermal applications.Dosage treatment may be a single dose schedule or a multiple doseschedule. The vaccine may be administered in conjunction with otherimmunoregulatory agents.

As an alternative to protein-based vaccines, DNA vaccination may beemployed [eg. Robinson & Torres (1997) Seminars in Immunology 9:271-283;Donnelly et al. (1997) Annu Rev Immunol 15:617-648; see later herein].

Gene Delivery Vehicles

Gene therapy vehicles for delivery of constructs including a codingsequence of a therapeutic of the invention, to be delivered to themammal for expression in the mammal, can be administered either locallyor systemically. These constructs can utilize viral or non-viral vectorapproaches in in vivo or ex vivo modality. Expression of such codingsequence can be induced using endogenous mammalian or heterologouspromoters. Expression of the coding sequence in vivo can be eitherconstitutive or regulated.

The invention includes gene delivery vehicles capable of expressing thecontemplated nucleic acid sequences. The gene delivery vehicle ispreferably a viral vector and, more preferably, a retroviral,adenoviral, adeno-associated viral (AAV), herpes viral, or alphavirusvector. The viral vector can also be an astrovirus, coronavirus,orthomyxovirus, papovavirus, paramyxovirus, parvovirus, picornavirus,poxvirus, or togavirus viral vector. See generally, Jolly (1994) CancerGene Therapy 1:51-64; Kimura (1994) Human Gene Therapy 5:845-852;Connelly (1995) Human Gene Therapy 6:185-193; and Kaplitt (1994) NatureGenetics 6:148-153.

Retroviral vectors are well known in the art and we contemplate that anyretroviral gene therapy vector is employable in the invention, includingB, C and D type retroviruses, xenotropic retroviruses (for example,NZB-X1, NZB-X2 and NZB9-1 (see O'Neill (1985) J. Virol. 53:160)polytropic retroviruses eg. MCF and MCF-MLV (see Kelly (1983) J. Virol.45:291), spumaviruses and lentiviruses. See RNA Tumor Viruses, SecondEdition, Cold Spring Harbor Laboratory, 1985.

Portions of the retroviral gene therapy vector may be derived fromdifferent retroviruses. For example, retrovector LTRs may be derivedfrom a Murine Sarcoma Virus, a tRNA binding site from a Rous SarcomaVirus, a packaging signal from a Murine Leukemia Virus, and an origin ofsecond strand synthesis from an Avian Leukosis Virus.

These recombinant retroviral vectors may be used to generatetransduction competent retroviral vector particles by introducing theminto appropriate packaging cell lines (see U.S. Pat. No. 5,591,624).Retrovirus vectors can be constructed for site-specific integration intohost cell DNA by incorporation of a chimeric integrase enzyme into theretroviral particle (see WO96/37626). It is preferable that therecombinant viral vector is a replication defective recombinant virus.

Packaging cell lines suitable for use with the above-describedretrovirus vectors are well known in the art, are readily prepared (seeWO95/30763 and WO92/05266), and can be used to create producer celllines (also termed vector cell lines or “VCLs”) for the production ofrecombinant vector particles. Preferably, the packaging cell lines aremade from human parent cells (eg. HT1080 cells) or mink parent celllines, which eliminates inactivation in human serum.

Preferred retroviruses for the construction of retroviral gene therapyvectors include Avian Leukosis Virus, Bovine Leukemia, Virus, MurineLeukemia Virus, Mink-Cell Focus-Inducing Virus, Murine Sarcoma Virus,Reticuloendotheliosis Virus and Rous Sarcoma Virus. Particularlypreferred Murine Leukemia Viruses include 4070A and 1504A (Hartley andRowe (1976) J Virol 19:19-25), Abelson (ATCC No. VR-999), Friend (ATCCNo. VR-245), Graffi, Gross (ATCC Nol VR-590), Kirsten, Harvey SarcomaVirus and Rauscher (ATCC No. VR-998) and Moloney Murine Leukemia Virus(ATCC No. VR-190). Such retroviruses may be obtained from depositoriesor collections such as the American Type Culture Collection (“ATCC”) inRockville, Md. or isolated from known sources using commonly availabletechniques.

Exemplary known retroviral gene therapy vectors employable in thisinvention include those described in patent applications GB2200651,EP0415731, EP0345242, EP0334301, WO89/02468; WO89/05349, WO89/09271,WO90/02806, WO90/07936, WO94/03622, WO93/25698, WO93/25234, WO93/11230,WO93/10218, WO91/02805, WO91/02825, WO95/07994, U.S. Pat. No. 5,219,740,U.S. Pat. No. 4,405,712, U.S. Pat. No. 4,861,719, U.S. Pat. No.4,980,289, U.S. Pat. No. 4,777,127, U.S. Pat. No. 5,591,624. See alsoVile (1993) Cancer Res 53:3860-3864; Vile (1993) Cancer Res 53:962-967;Ram (1993) Cancer Res 53 (1993) 83-88; Takamiya (1992) J Neurosci Res33:493-503; Baba (1993) J Neurosurg 79:729-735; Mann (1983) Cell 33:153;Cane (1984) Proc Natl Acad Sci 81:6349; and Miller (1990) Human GeneTherapy 1.

Human adenoviral gene therapy vectors are also known in the art andemployable in this invention. See, for example, Berkner (1988)Biolechniques 6:616 and Rosenfeld (1991) Science 252:431, andWO93/07283, WO93/06223, and WO93/07282. Exemplary known adenoviral genetherapy vectors employable in this invention include those described inthe above referenced documents and in WO94/12649, WO93/03769,WO93/19191, WO94/28938, WO95/11984, WO95/00655, WO95/27071, WO95/29993,WO95/34671, WO96/05320, WO94/08026, WO94/11506, WO93/06223, WO94/24299,WO95/14102, WO95/24297, WO95/02697, WO94/28152, WO94/24299, WO95/09241,WO95/25807, WO95/05835, WO94/18922 and WO95/09654. Alternatively,administration of DNA linked to killed adenovirus as described in Curiel(1992) Hum. Gene Ther. 3:147-154 may be employed. The gene deliveryvehicles of the invention also include adenovirus associated virus (AAV)vectors. Leading and preferred examples of such vectors for use in thisinvention are the AAV-2 based vectors disclosed in Srivastava,WO93/09239. Most preferred AAV vectors comprise the two AAV invertedterminal repeats in which the native D-sequences are modified bysubstitution of nucleotides, such that at least 5 native nucleotides andup to 18 native nucleotides, preferably at least 10 native nucleotidesup to 18 native nucleotides, most preferably 10 native nucleotides areretained and the remaining nucleotides of the D-sequence are deleted orreplaced with non-native nucleotides. The native D-sequences of the AAVinverted terminal repeats are sequences of 20 consecutive nucleotides ineach AAV inverted terminal repeat (ie. there is one sequence at eachend) which are not involved in HP formation. The non-native replacementnucleotide may be any nucleotide other than the nucleotide found in thenative D-sequence in the same position. Other employable exemplary AAVvectors are pWP-19, pWN-1, both of which are disclosed in Nahreini(1993) Gene 124:257-262. Another example of such an AAV vector ispsub201 (see Samulski (1987) J. Virol. 61:3096). Another exemplary AAVvector is the Double-D ITR vector. Construction of the Double-D ITRvector is disclosed in U.S. Pat. No. 5,478,745. Still other vectors arethose disclosed in Carter U.S. Pat. No. 4,797,368 and Muzyczka U.S. Pat.No. 5,139,941, Chartejee U.S. Pat. No. 5,474,935, and Kotin WO94/288157.Yet a further example of an AAV vector employable in this invention isSSV9AFABTKneo, which contains the AFP enhancer and albumin promoter anddirects expression predominantly in the liver. Its structure andconstruction are disclosed in Su (1996) Human Gene Therapy 7:463-470.Additional AAV gene therapy vectors are described in U.S. Pat. No.5,354,678, U.S. Pat. No. 5,173,414, U.S. Pat. No. 5,139,941, and U.S.Pat. No. 5,252,479.

The gene therapy vectors of the invention also include herpes vectors.Leading and preferred examples are herpes simplex virus vectorscontaining a sequence encoding a thymidine kinase polypeptide such asthose disclosed in U.S. Pat. No. 5,288,641 and EP0176170 (Roizman).Additional exemplary herpes simplex virus vectors include HFEM/ICP6-LacZdisclosed in WO95/04139 (Wistar Institute), pHSVlac described in Geller(1988) Science 241:1667-1669 and in WO90/09441 and WO92/07945, HSVUs3::pgC-lacZ described in Fink (1992) Human Gene Therapy 3:11-19 andHSV 7134, 2 RH 105 and GAL4 described in EP 0453242 (Breakefield), andthose deposited with the ATCC as accession numbers ATCC VR-977 and ATCCVR-260.

Also contemplated are alpha virus gene therapy vectors that can beemployed in this invention. Preferred alpha virus vectors are Sindbisviruses vectors. Togaviruses, Semliki Forest virus (ATCC VR-67; ATCCVR-1247), Middleberg virus (ATCC VR-370), Ross River virus (ATCC VR-373;ATCC VR-1246), Venezuelan equine encephalitis virus (ATCC VR923; ATCCVR-1250; ATCC VR-1249; ATCC VR-532), and those described in U.S. Pat.Nos. 5,091,309, 5,217,879, and WO92/10578. More particularly, thosealpha virus vectors described in U.S. Ser. No. 08/405,627, filed Mar.15, 1995, WO94/21792, WO92/10578, WO95/07994, U.S. Pat. No. 5,091,309and U.S. Pat. No. 5,217,879 are employable. Such alpha viruses may beobtained from depositories or collections such as the ATCC in Rockville,Md. or isolated from known sources using commonly available techniques.Preferably, alphavirus vectors with reduced cytotoxicity are used (seeU.S. Ser. No. 08/679,640).

DNA vector systems such as eukaryotic layered expression systems arealso useful for expressing the nucleic acids of the invention. SeeWO95/07994 for a detailed description of eukaryotic layered expressionsystems. Preferably, the eukaryotic layered expression systems of theinvention are derived from alphavirus vectors and most preferably fromSindbis viral vectors.

Other viral vectors suitable for use in the present invention includethose derived from poliovirus, for example ATCC VR-58 and thosedescribed in Evans, Nature 339 (1989) 385 and Sabin (1973) J. Biol.Standardization 1:115; rhinovirus, for example ATCC VR-1110 and thosedescribed in Arnold (1990) J Cell Biochem L401; pox viruses such ascanary pox virus or vaccinia virus, for example ATCC VR-111 and ATCCVR-2010 and those described in Fisher-Hoch (1989) Proc Natl Acad Sci86:317; Flexner (1989) Ann NY Acad Sci 569:86, Flexner (1990) Vaccine8:17; in U.S. Pat. No. 4,603,112 and U.S. Pat. No. 4,769,330 andWO89/01973; SV40 virus, for example ATCC VR-305 and those described inMulligan (1979) Nature 277:108 and Madzak (1992) J Gen Virol 73:1533;influenza virus, for example ATCC VR-797 and recombinant influenzaviruses made employing reverse genetics techniques as described in U.S.Pat. No. 5,166,057 and in Enami (1990) Proc Natl Acad Sci 87:3802-3805;Enami & Palese (1991) J Virol 65:2711-2713 and Luytjes (1989) Cell59:110, (see also McMichael (1983) NEJ Med 309:13, and Yap (1978) Nature273:238 and Nature (1979) 277:108); human immunodeficiency virus asdescribed in EP-0386882 and in Buchschacher (1992) J. Virol. 66:2731;measles virus, for example ATCC VR-67 and VR-1247 and those described inEP-0440219; Aura virus, for example ATCC VR-368; Bebaru virus, forexample ATCC VR-600 and ATCC VR-1240; Cabassou virus, for example ATCCVR-922; Chikungunya virus, for example ATCC VR-64 and ATCC VR-1241; FortMorgan Virus, for example ATCC VR-924; Getah virus, for example ATCCVR-369 and ATCC VR-1243; Kyzylagach virus, for example ATCC VR-927;Mayaro virus, for example ATCC VR-66; Mucambo virus, for example ATCCVR-580 and ATCC VR-1244, Ndumu virus, for example ATCC VR-371; Pixunavirus, for example ATCC VR-372 and ATCC VR-1245; Tonate virus, forexample ATCC VR-925; Triniti virus, for example ATCC VR-469; Una virus,for example ATCC VR-374; Whataroa virus, for example ATCC VR-926;Y-62-33 virus, for example ATCC VR-375; O'Nyong virus, Easternencephalitis virus, for example ATCC VR-65 and ATCC VR-1242; Westernencephalitis virus, for example ATCC VR-70, ATCC VR-1251, ATCC VR-622and ATCC VR-1252; and coronavirus, for example ATCC VR-740 and thosedescribed in Hamre (1966) Proc Soc Exp Biol Med 121:190.

Delivery of the compositions of this invention into cells is not limitedto the above mentioned viral vectors. Other delivery methods and mediamay be employed such as, for example, nucleic acid expression vectors,polycationic condensed DNA linked or unlinked to killed adenovirusalone, for example see U.S. Ser. No. 08/366,787, filed Dec. 30, 1994 andCuriel (1992) Hum Gene Ther 3:147-154 ligand linked DNA, for example seeWu (1989) J Biol Chem 264:16985-16987, eucaryotic cell delivery vehiclescells, for example see U.S. Ser. No. 08/240,030, filed May 9, 1994, andU.S. Ser. No. 08/404,796, deposition of photopolymerized hydrogelmaterials, hand-held gene transfer particle gun, as described in U.S.Pat. No. 5,149,655, ionizing radiation as described in U.S. Pat. No.5,206,152 and in WO92/11033, nucleic charge neutralization or fusionwith cell membranes. Additional approaches are described in Philip(1994) Mol Cell Biol 14:2411-2418 and in Woffendin (1994) Proc Natl AcadSci 91:1581-1585.

Particle mediated gene transfer may be employed, for example see U.S.Ser. No. 60/023,867. Briefly, the sequence can be inserted intoconventional vectors that contain conventional control sequences forhigh level expression, and then incubated with synthetic gene transfermolecules such as polymeric DNA-binding cations like polylysine,protamine, and albumin, linked to cell targeting ligands such asasialoorosomucoid, as described in Wu & Wu (1987) J. Biol. Chem.262:4429-4432, insulin as described in Hucked (1990) Biochem Pharmacol40:253-263, galactose as described in Plank (1992) Bioconjugate Chem3:533-539, lactose or transferrin.

Naked DNA may also be employed. Exemplary naked DNA introduction methodsare described in WO 90/11092 and U.S. Pat. No. 5,580,859. Uptakeefficiency may be improved using biodegradable latex beads. DNA coatedlatex beads are efficiently transported into cells after endocytosisinitiation by the beads. The method may be improved further by treatmentof the beads to increase hydrophobicity and thereby facilitatedisruption of the endosome and release of the DNA into the cytoplasm.

Liposomes that can act as gene delivery vehicles are described in U.S.Pat. No. 5,422,120, WO95/13796, WO94/23697, WO91/14445 and EP-524,968.As described in U.S. Ser. No. 60/023,867, on non-viral delivery, thenucleic acid sequences encoding a polypeptide can be inserted intoconventional vectors that contain conventional control sequences forhigh level expression, and then be incubated with synthetic genetransfer molecules such as polymeric DNA-binding cations likepolylysine, protamine, and albumin, linked to cell targeting ligandssuch as asialoorosomucoid, insulin, galactose, lactose, or transferrin.Other delivery systems include the use of liposomes to encapsulate DNAcomprising the gene under the control of a variety of tissue-specific orubiquitously-active promoters. Further non-viral delivery suitable foruse includes mechanical delivery systems such as the approach describedin Woffendin et al (1994) Proc. Natl. Acad. Sci. USA 91(24):11581-11585.Moreover, the coding sequence and the product of expression of such canbe delivered through deposition of photopolymerized hydrogel materials.Other conventional methods for gene delivery that can be used fordelivery of the coding sequence include, for example, use of hand-heldgene transfer particle gun, as described in U.S. Pat. No. 5,149,655; useof ionizing radiation for activating transferred gene, as described inU.S. Pat. No. 5,206,152 and WO92/11033

Exemplary liposome and polycationic gene delivery vehicles are thosedescribed in U.S. Pat. Nos. 5,422,120 and 4,762,915; in WO 95/13796;WO94/23697; and WO91/14445; in EP-0524968; and in Stryer, Biochemistry,pages 236-240 (1975) W.H. Freeman, San Francisco; Szoka (1980) BiochemBiophys Acta 600:1; Bayer (1979) Biochem Biophys Acta 550:464; Rivnay(1987) Meth Enzymol 149:119; Wang (1987) Proc Natl Acad Sci 84:7851;Plant (1989) Anal Biochem 176:420.

A polynucleotide composition can comprises therapeutically effectiveamount of a gene therapy vehicle, as the term is defined above. Forpurposes of the present invention, an effective dose will be from about0.01 mg/kg to 50 mg/kg or 0.05 mg/kg to about 10 mg/kg of the DNAconstructs in the individual to which it is administered.

Delivery Methods

Once formulated, the polynucleotide compositions of the invention can beadministered (1) directly to the subject; (2) delivered ex vivo, tocells derived from the subject; or (3) in vitro for expression ofrecombinant proteins. The subjects to be treated can be mammals orbirds. Also, human subjects can be treated.

Direct delivery of the compositions will generally be accomplished byinjection, either subcutaneously, intraperitoneally, intravenously orintramuscularly or delivered to the interstitial space of a tissue. Thecompositions can also be administered into a lesion. Other modes ofadministration include oral and pulmonary administration, suppositories,and transdermal or transcutaneous applications (eg. see WO98/20734),needles, and gene guns or hyposprays. Dosage treatment may be a singledose schedule or a multiple dose schedule.

Methods for the ex vivo delivery and reimplantation of transformed cellsinto a subject are known in the art and described in eg. WO93/14778.Examples of cells useful in ex vivo applications include, for example,stem cells, particularly hematopoetic, lymph cells, macrophages,dendritic cells, or tumor cells.

Generally, delivery of nucleic acids for both ex vivo and in vitroapplications can be accomplished by the following procedures, forexample, dextran-mediated transfection, calcium phosphate precipitation,polybrene mediated transfection, protoplast fusion, electroporation,encapsulation of the polynucleotide(s) in liposomes, and directmicroinjection of the DNA into nuclei, all well known in the art.

Polynucleotide and Polypeptide Pharmaceutical Compositions

In addition to the pharmaceutically acceptable carriers and saltsdescribed above, the following additional agents can be used withpolynucleotide and/or polypeptide compositions.

A. Polypeptides

One example are polypeptides which include, without limitation:asioloorosomucoid (ASOR); transferrin; asialoglycoproteins; antibodies;antibody fragments; ferritin; interleukins; interferons, granulocyte,macrophage colony stimulating factor (GM-CSF), granulocyte colonystimulating factor (G-CSF), macrophage colony stimulating factor(M-CSF), stem cell factor and erythropoietin. Viral antigens, such asenvelope proteins, can also be used. Also, proteins from other invasiveorganisms, such as the 17 amino acid peptide from the circumsporozoiteprotein of plasmodium falciparum known as RII.

B. Hormones Vitamins, etc.

Other groups that can be included are, for example: hormones, steroids,androgens, estrogens, thyroid hormone, or vitamins, folic acid.

C. Polyalkylenes, Polysaccharides, etc.

Also, polyalkylene glycol can be included with the desiredpolynucleotides/polypeptides. In a preferred embodiment, thepolyalkylene glycol is polyethylene glycol. In addition, mono-, di-, orpolysaccharides can be included. In a preferred embodiment of thisaspect, the polysaccharide is dextran or DEAE-dextran. Also, chitosanand poly(lactide-co-glycolide)

D. Lipids and Liposomes

The desired polynucleotide/polypeptide can also be encapsulated inlipids or packaged in liposomes prior to delivery to the subject or tocells derived therefrom.

Lipid encapsulation is generally accomplished using liposomes which areable to stably bind or entrap and retain nucleic acid. The ratio ofcondensed polynucleotide to lipid preparation can vary but willgenerally be around 1:1 (mg DNA:micromoles lipid), or more of lipid. Fora review of the use of liposomes as carriers for delivery of nucleicacids, see, Hug and Sleight (1991) Biochim. Biophys. Acta. 1097:1-17;Straubinger (1983) Meth. Enzymol. 101:512-527.

Liposomal preparations for use in the present invention include cationic(positively charged), anionic (negatively charged) and neutralpreparations. Cationic liposomes have been shown to mediateintracellular delivery of plasmid DNA (Felgner (1987) Proc. Natl. Acad.Sci. USA 84:7413-7416); mRNA (Malone (1989) Proc. Natl. Acad. Sci. USA86:6077-6081); and purified transcription factors (Debs (1990) J. Biol.Chem. 265:10189-10192), in functional form.

Cationic liposomes are readily available. For example,N[1-2,3-dioleyloxy)propyl]-N,N,N-triethylammonium (DOTMA) liposomes areavailable under the trademark Lipofectin, from GIBCO BRL, Grand Island,N.Y. (See, also, Felgner supra). Other commercially available liposomesinclude transfectace (DDAB/DOPE) and DOTAP/DOPE (Boerhinger). Othercationic liposomes can be prepared from readily available materialsusing techniques well known in the art. See, eg. Szoka (1978) Proc.Natl. Acad. Sci. USA 75:4194-4198; WO90/11092 for a description of thesynthesis of DOTAP (1,2-bis(oleoyloxy)-3-(trimethylammonio)propane)liposomes.

Similarly, anionic and neutral liposomes are readily available, such asfrom Avanti Polar Lipids (Birmingham, Ala.), or can be easily preparedusing readily available materials. Such materials include phosphatidylcholine, cholesterol, phosphatidyl ethanolamine, dioleoylphosphatidylcholine (DOPC), dioleoylphosphatidyl glycerol (DOPG),dioleoylphoshatidyl ethanolamine (DOPE), among others. These materialscan also be mixed with the DOTMA and DOTAP starting materials inappropriate ratios. Methods for making liposomes using these materialsare well known in the art.

The liposomes can comprise multilammelar vesicles (MLVs), smallunilamellar vesicles (SUVs), or large unilamellar vesicles (LUVs). Thevarious liposome-nucleic acid complexes are prepared using methods knownin the art. See eg. Straubinger (1983) Meth. Immunol. 101:512-527; Szoka(1978) Proc. Natl. Acad. Sci. USA 75:4194-4198; Papahadjopoulos (1975)Biochim. Biophys. Acta 394:483; Wilson (1979) Cell 17:77); Deamer &Bangham (1976) Biochim. Biophys. Acta 443:629; Ostro (1977) Biochem.Biophys. Res. Commun. 76:836; Fraley (1979) Proc. Natl. Acad. Sci. USA76:3348); Enoch & Strittmatter (1979) Proc. Natl. Acad. Sci. USA 76:145;Fraley (1980) J. Biol. Chem. (1980) 255:10431; Szoka & Papahadjopoulos(1978) Proc. Natl. Acad. Sci. USA 75:145; and Schaefer-Ridder (1982)Science 215:166.

E. Lipoproteins

In addition, lipoproteins can be included with thepolynucleotide/polypeptide to be delivered. Examples of lipoproteins tobe utilized include: chylomicrons, HDL, IDL, LDL, and VLDL. Mutants,fragments, or fusions of these proteins can also be used. Also,modifications of naturally occurring lipoproteins can be used, such asacetylated LDL. These lipoproteins can target the delivery ofpolynucleotides to cells expressing lipoprotein receptors. Preferably,if lipoproteins are including with the polynucleotide to be delivered,no other targeting ligand is included in the composition.

Naturally occurring lipoproteins comprise a lipid and a protein portion.The protein portion are known as apoproteins. At the present,apoproteins A, B, C, D, and E have been isolated and identified. Atleast two of these contain several proteins, designated by Romannumerals, AI, AII, AIV; CI, CII, CIII.

A lipoprotein can comprise more than one apoprotein. For example,naturally occurring chylomicrons comprises of A, B, C, and E, over timethese lipoproteins lose A and acquire C and E apoproteins. VLDLcomprises A, B, C, and E apoproteins, LDL comprises apoprotein B; andHDL comprises apoproteins A, C, and E.

The amino acid of these apoproteins are known and are described in, forexample, Breslow (1985) Annu Rev. Biochem 54:699; Law (1986) Adv. ExpMed. Biol. 151:162; Chen (1986) J Biol Chem 261:12918; Kane (1980) ProcNatl Acad Sci USA 77:2465; and Utermann (1984) Hum Genet. 65:232.

Lipoproteins contain a variety of lipids including, triglycerides,cholesterol (free and esters), and phospholipids. The composition of thelipids varies in naturally occurring lipoproteins. For example,chylomicrons comprise mainly triglycerides. A more detailed descriptionof the lipid content of naturally occurring lipoproteins can be found,for example, in Meth. Enzymol. 128 (1986). The composition of the lipidsare chosen to aid in conformation of the apoprotein for receptor bindingactivity. The composition of lipids can also be chosen to facilitatehydrophobic interaction and association with the polynucleotide bindingmolecule.

Naturally occurring lipoproteins can be isolated from serum byultracentrifugation, for instance. Such methods are described in Meth.Enzymol. (supra); Pitas (1980) J. Biochem. 255:5454-5460 and Mahey(1979) J. Clin. Invest 64:743-750. Lipoproteins can also be produced byin vitro or recombinant methods by expression of the apoprotein genes ina desired host cell. See, for example, Atkinson (1986) Annu Rev BiophysChem 15:403 and Radding (1958) Biochim Biophys Acta 30: 443.Lipoproteins can also be purchased from commercial suppliers, such asBiomedical Technologies, Inc., Stoughton, Mass., USA. Furtherdescription of lipoproteins can be found in WO98/06437.

F. Polycationic Agents

Polycationic agents can be included, with or without lipoprotein, in acomposition with the desired polynucleotide/polypeptide to be delivered.

Polycationic agents, typically, exhibit a net positive charge atphysiological relevant pH and are capable of neutralizing the electricalcharge of nucleic acids to facilitate delivery to a desired location.These agents have both in vitro, ex vivo, and in vivo applications.Polycationic agents can be used to deliver nucleic acids to a livingsubject either intramuscularly, subcutaneously, etc.

The following are examples of useful polypeptides as polycationicagents: polylysine, polyarginine, polyornithine, and protamine. Otherexamples include histones, protamines, human serum albumin, DNA bindingproteins, non-histone chromosomal proteins, coat proteins from DNAviruses, such as (X174, transcriptional factors also contain domainsthat bind DNA and therefore may be useful as nucleic aid condensingagents. Briefly, transcriptional factors such as C/CEBP, c-jun, c-fos,AP-1, AP-2, AP-3, CPF, Prot-1, Sp-1, Oct-1, Oct-2, CREP, and TFIIDcontain basic domains that bind DNA sequences.

Organic polycationic agents include: spermine, spermidine, andpurtrescine.

The dimensions and of the physical properties of a polycationic agentcan be extrapolated from the list above, to construct other polypeptidepolycationic agents or to produce synthetic polycationic agents.

Synthetic polycationic agents which are useful include, for example,DEAE-dextran, polybrene. Lipofectin™, and LipofectAMINE™ are monomersthat form polycationic complexes when combined withpolynucleotides/polypeptides.

Immunodiagnostic Assays

Neisserial antigens of the invention can be used in immunoassays todetect antibody levels (or, conversely, anti-Neisserial antibodies canbe used to detect antigen levels). Immunoassays based on well defined,recombinant antigens can be developed to replace invasive diagnosticsmethods. Antibodies to Neisserial proteins within biological samples,including for example, blood or serum samples, can be detected. Designof the immunoassays is subject to a great deal of variation, and avariety of these are known in the art. Protocols for the immunoassay maybe based, for example, upon competition, or direct reaction, or sandwichtype assays. Protocols may also, for example, use solid supports, or maybe by immunoprecipitation. Most assays involve the use of labeledantibody or polypeptide; the labels may be, for example, fluorescent,chemiluminescent, radioactive, or dye molecules. Assays which amplifythe signals from the probe are also known; examples of which are assayswhich utilize biotin and avidin, and enzyme-labeled and mediatedimmunoassays, such as ELISA assays.

Kits suitable for immunodiagnosis and containing the appropriate labeledreagents are constructed by packaging the appropriate materials,including the compositions of the invention, in suitable containers,along with the remaining reagents and materials (for example, suitablebuffers, salt solutions, etc.) required for the conduct of the assay, aswell as suitable set of assay instructions.

Nucleic Acid Hybridisation

“Hybridization” refers to the association of two nucleic acid sequencesto one another by hydrogen bonding. Typically, one sequence will befixed to a solid support and the other will be free in solution. Then,the two sequences will be placed in contact with one another underconditions that favor hydrogen bonding. Factors that affect this bondinginclude: the type and volume of solvent; reaction temperature; time ofhybridization; agitation; agents to block the non-specific attachment ofthe liquid phase sequence to the solid support (Denhardt's reagent orBLOTTO); concentration of the sequences; use of compounds to increasethe rate of association of sequences (dextran sulfate or polyethyleneglycol); and the stringency of the washing conditions followinghybridization. See Sambrook et al. [supra] Volume 2, chapter 9, pages9.47 to 9.57.

“Stringency” refers to conditions in a hybridization reaction that favorassociation of very similar sequences over sequences that differ. Forexample, the combination of temperature and salt concentration should bechosen that is approximately 120 to 200° C. below the calculated Tm ofthe hybrid under study. The temperature and salt conditions can often bedetermined empirically in preliminary experiments in which samples ofgenomic DNA immobilized on filters are hybridized to the sequence ofinterest and then washed under conditions of different stringencies. SeeSambrook et al. at page 9.50.

Variables to consider when performing, for example, a Southern blot are(1) the complexity of the DNA being blotted and (2) the homology betweenthe probe and the sequences being detected. The total amount of thefragment(s) to be studied can vary a magnitude of 10, from 0.1 to 1 μgfor a plasmid or phage digest to 10⁻⁹ to 10⁻⁸ g for a single copy genein a highly complex eukaryotic genome. For lower complexitypolynucleotides, substantially shorter blotting, hybridization, andexposure times, a smaller amount of starting polynucleotides, and lowerspecific activity of probes can be used. For example, a single-copyyeast gene can be detected with an exposure time of only 1 hour startingwith 1 μg of yeast DNA, blotting for two hours, and hybridizing for 4-8hours with a probe of 10⁸ cpm/μg. For a single-copy mammalian gene aconservative approach would start with 10 μg of DNA, blot overnight, andhybridize overnight in the presence of 10% dextran sulfate using a probeof greater than 10⁸ cpm/μg, resulting in an exposure time of ˜24 hours.

Several factors can affect the melting temperature (Tm) of a DNA-DNAhybrid between the probe and the fragment of interest, and consequently,the appropriate conditions for hybridization and washing. In many casesthe probe is not 100% homologous to the fragment. Other commonlyencountered variables include the length and total G+C content of thehybridizing sequences and the ionic strength and formamide content ofthe hybridization buffer. The effects of all of these factors can beapproximated by a single equation:

Tm=81+16.6(log₁₀ Ci)+0.4[%(G+C)]−0.6(% formamide)−600/n−1.5(% mismatch).

where Ci is the salt concentration (monovalent ions) and n is the lengthof the hybrid in base pairs (slightly modified from Meinkoth & Wahl(1984) Anal. Biochem. 138: 267-284).

In designing a hybridization experiment, some factors affecting nucleicacid hybridization can be conveniently altered. The temperature of thehybridization and washes and the salt concentration during the washesare the simplest to adjust. As the temperature of the hybridizationincreases (ie. stringency), it becomes less likely for hybridization tooccur between strands that are nonhomologous, and as a result,background decreases. If the radiolabeled probe is not completelyhomologous with the immobilized fragment (as is frequently the case ingene family and interspecies hybridization experiments), thehybridization temperature must be reduced, and background will increase.The temperature of the washes affects the intensity of the hybridizingband and the degree of background in a similar manner. The stringency ofthe washes is also increased with decreasing salt concentrations.

In general, convenient hybridization temperatures in the presence of 50%formamide are 42° C. for a probe with is 95% to 100% homologous to thetarget fragment, 37° C. for 90% to 95% homology, and 32° C. for 85% to90% homology. For lower homologies, formamide content should be loweredand temperature adjusted accordingly, using the equation above. If thehomology between the probe and the target fragment are not known, thesimplest approach is to start with both hybridization and washconditions which are nonstringent. If non-specific bands or highbackground are observed after autoradiography, the filter can be washedat high stringency and reexposed. If the time required for exposuremakes this approach impractical, several hybridization and/or washingstringencies should be tested in parallel.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1D to 7A-7D show BOXSHADE-rendered alignments of (1) ORF40 (SEQID NOS:1-21) (2) ORF4 (SEQ ID NOS:22-53) (3) 225 (SEQ ID NOS:54-87) (4)235 (SEQ ID NOS:88-118) (5) 287 (SEQ ID NOS:119-124) (6) 519 (SEQ IDNOS:125-146) (7) 919 (SEQ ID NOS:147-181).

Conserved amino acids have a solid background.

FIG. 8 shows a phylogenetic tree.

FIG. 9A illustrates amino acid sequence variability within N.meningitidis for ORF4, ORF40, 225, 235, 287, 519, and 919. Thesesequences were used to construct the phylogenetic tree shown in FIG. 9B.

FIGS. 10A-10B to 19 show BOXSHADE-rendered alignments of (10) ORF4 (SEQID NOS:22-53) (11) ORF40 (SEQ ID NOS:1-21) (12) ORF46 (SEQ IDNOS:182-187) (13) 225 (SEQ ID NOS: 54-87) (14) 235 (SEQ ID NOS: 88-118)(15) 287 (SEQ ID NOS: 119-124) (16) 519 (SEQ ID NOS: 125-146) (17) 726(SEQ ID NOS:188-195) (18) 919 (SEQ ID NOS: 147-181) (19) 953 (SEQ IDNOS:196-203).

FIGS. 20A-20E shows Western blots for ORF4, 225, 235, 519 and 919.

EXAMPLES Example 1

Example 1 of WO99/36544 discloses the cloning and expression of aNeisserial protein referred to as “ORF40”. Protein and DNA sequencesfrom serogroup A and B N. meningitidis are disclosed, and the completeprotein sequences show 83.7% identity over 601 aa overlap.

ORF40 was sequenced for a reference population of 21 strains of N.meningitidis:

Identification number Strain Reference Group B zn02_1 BZ198 Seiler etal.(1996) zn03_1 NG3/88 Seiler et al.(1996) zn04_1 297-0 Seiler etal.(1996) zn06_1 BZ147 Seiler et al.(1996) zn07_1 BZ169 Seiler etal.(1996) zn08_1 528 Seiler et al.(1996) zn10_1 BZ133 Seiler etal.(1996) zh11_1ass NGE31 Seiler et al.(1996) zn14_1 NGH38 Seiler etal.(1996) zn16_1 NGH15 Seiler et al.(1996) zn18_1 BZ232 Seiler etal.(1996) zn19_1 BZ83 Seiler et al.(1996) zn20_1 44/76 Seiler etal.(1996) zn21_1 MC58 Virji et al. (1992) Group A zn22_1 205900 ChironSpA zn23_1 F6124 Chiron SpA z2491_1 Z2491 Maiden et al.(1998) Group Czn24_1 90/18311 Chiron SpA zn25_1ass 93/4286 Chiron SpA Others zn28_1ass860800 (group Y) Maiden et al.(1998) zn29_1ass E32 (group Z) Maiden etal.(1998)

An alignment of these 21 sequences is shown in FIGS. 1A-1D. Stretches ofconserved amino acids are evident. The first 17 amino acids, forinstance, are conserved (MNKIYRIIWNSALNAWV SEQ ID NO:204), although theserine at residue 11 is not present in 100% of Neisseria. This isfollowed by an amino acid which is not conserved, which is in turnfollowed by a stretch of 16 conserved amino acids (VSELTRNHTKRASATV SEQID NO:205). The C-terminal of the protein consists of 116 conservedamino acids.

The conserved regions identified in this example confirm that fragmentsof the full-length ORF40 protein are suitable as multi-specific vaccinesor diagnostic reagents.

ORF40 was re-sequenced for 31 strains in total, and the sequences werealigned. The results are shown in FIGS. 11A-11D.

Conserved regions of particular interest are:

(SEQ ID NO:204) MNKIYRIIWNSALNAWV (SEQ ID NO:205) VSELTRNHTKRASATV (SEQID NO:206) AVLATLL (SEQ ID NO:207) TLKAGDNLKIKQ (SEQ ID NO:208)FTYSLKKDLTDLTSV (SEQ ID NO:209) TEKLSFGANG (SEQ ID NO:210)KVNITSDTKGLNFAKETAGTNGD (SEQ ID NO:211) TVHLNGIGSTLTDTL (SEQ ID NO:212)RAAS (V/I) KDVLNAGWNIKGVK (SEQ ID NO:213)NVDFVRTYDTVEFLSADTKTTTVNVESKDNGKKTEVKIGAKTSVIKEKDG KLVTGK (SEQ IDNO:214) KGENGSSTDEGEGLVTAKEVIDAVNKAGWRMKTTTANGQTGQADKFETVT SGT (SEQ IDNO:215) GTTATVSKDDQGNITV (SEQ ID NO:216)YDVNVGDALNVNQLQNSGWNLDSKAVAGSSGKVISGNVSPSKGKNDETVNINAGNNIEITRNGKNIDIATSM (SEQ ID NO:217) PQFSSVSLGAGADAPTLSVD (SEQ IDNO:218) NKPVRITNVAPGVKEGDVTNVAQLKGVAQNLNNRIDNVDGNARAGIAQAIATAGLVQAYLPGKSMMAIGGGTYRGEAGYAIGYSSISDGGNWIIKGTASG NSRGHFGASASVGYQW.

Example 2

Example 26 of WO99/24578 discloses the cloning and expression of aNeisserial protein referred to as “ORF4”. Protein and DNA sequences fromserogroup A and B N. meningitidis are disclosed, along with sequencesfrom N. gonorrhoeae. The identity between the sequences at an amino acidlevel are:

N. meningitidis A N. gonorrhoeae N. meningitidis B 99.7% over 287 aa97.6% over 288 aa

ORF4 was sequenced for a reference population of 32 strains ofNeisseria:

Identification number Strain Reference Group B zv01_4 NG6/88 Seiler etal. (1996) zv02_4 BZ198 Seiler et al. (1996) zv03_4ass NG3/88 Seiler etal. (1996) zv04_4 297-0 Seiler et al. (1996) zv05_4 1000 Seiler et al.(1996) zv06_4 BZ147 Seiler et al. (1996) zv07_4 BZ169 Seiler et al.(1996) zv08_4 528 Seiler et al. (1996) zv09_4 NGP165 Seiler et al.(1996) zv10_4 BZ133 Seiler et al. (1996) zv11_4 NGE31 Seiler et al.(1996) zv12_4ass NGF26 Seiler et al. (1996) zv13_4 NGE28 Seiler et al.(1996) zv15_4′ SWZ107 Seiler et al. (1996) zv16_4 NGH15 Seiler et al.(1996) zv17_4 NGH36 Seiler et al. (1996) zv18_4 BZ232 Seiler et al.(1996) zv19_4 BZ83 Seiler et al. (1996) zv20_4 44/76 Seiler et al.(1996) zv21_4 MC58 Virji et al. (1992) zv96_4 2996 Chiron SpA Group Azv22_4 205900 Chiron SpA z2491_4 Z2491 Maiden et al., 1998 Group Czv24_4 90/18311 Chiron SpA zv25_4 93/4286 Chiron SpA Others zv26_4assA22 (group W) Maiden et al. (1998) zv27_4 E26 (group X) Maiden et al.(1998) zv28_4 860800(group Y) Maiden et al. (1998) zv29_4 E32 (group Z)Maiden et al. (1998) N. gonorrhoeae zv32_4 Ng F62 Maiden et al. (1998)zv33_4 Ng SN4 R. Moxon fa1090_4 FA1090 Dempsey et al. (1991)

An alignment of the sequences generated using PILEUP is shown in FIGS.2A-2C. Stretches of conserved amino acids are evident. The first 34amino acids, for instance, are conserved, although the serine at residue26 is not present in 100% of Neisseria. The C-terminal of the proteinconsists of 228 conserved amino acids.

The conserved regions identified in this example confirm that fragmentsof the full-length ORF4 protein are suitable as multi-specific vaccinesor diagnostic reagents.

ORF4 was re-sequenced for 35 strains in total, and the sequences werealigned. The results are shown in FIGS. 10A-10B.

Conserved regions of particular interest are:

(SEQ ID NO:219) MKTFFKTLSAAALALILAACGGQKDSAPAASASAAADNGA (SEQ ID NO:220)KKEIVFGTTVGDFGDMVKE (SEQ ID NO:221)ELEKKGYTVKLVEFTDYVRPNLALAEGELDINVFQHKPYLDDFKKEHNLDITEVFQVPTAPLGLYPGKLKSLEEVKDGSTVSAPNDPSNFARVLVMLDELGWIKLKDGINPLTASKADIAENLKNIKIVELEAAQLPRSRADVDFAVVNGNYATSSGMKLTEALFQEPSFAYVNWSAVKTADKDSQWLKDVTEAYNSDAFKAYAHKRFEGYKSPAAWNEGAAK

Example 3

Example 16 of WO99/57280 discloses the cloning and expression of aNeisserial protein referred to as “225”. Protein and DNA sequences fromserogroup A and B N. meningitidis are disclosed, along with sequencesfrom N. gonorrhoeae.

225 has now been sequenced for a reference population of 34 strains ofNeisseria:

Identification number Strain Source reference Group B zo01_225 NG6/88Seiler et al., 1996 zo02_225 BZ198 Seiler et al., 1996 zo03_225 NG3/88Seiler et al., 1996 zo04_225 297-0 Seiler et al., 1996 zo05_225 1000Seiler et al., 1996 zo06_225 BZ147 Seiler et al., 1996 zo07_225 BZ169Seiler et al., 1996 zo08_225 528 Seiler et al., 1996 zo09_225 NGP165Seiler el al., 1996 zo10_225 BZ133 Seiler et al., 1996 zo11_225 NGE31Seiler et al., 1996 zo12_225 NGF26 Seiler et al., 1996 zo13_225 NGE28Seiler et al., 1996 zo14_225 NGH38 Seiler et al., 1996 zo15_225 SWZ107Seiler et al., 1996 zo16_225 NGH15 Seiler et al., 1996 zo17_225 NGH36Seiler et al., 1996 zo18_225 BZ232 Seiler et al., 1996 zo19_225 BZ83Seiler et al., 1996 zo20_225 44/76 Seiler et al., 1996 zo21_225 MC58Chiron SpA zo96_225 2996 Chiron SpA Group A zo22_225 205900 Chiron SpAzo23_225 F6124 Chiron SpA z2491 Z2491 Maiden et al., 1998 Group Czo24_225 90/18311 Chiron SpA zo25_225 93/4286 Chiron SpA Others zo26_225A22 (group W) Maiden et al., 1998 zo27_225 E26 (group X) Maiden et al.,1998 zo28_225 860800(group Y) Maiden et al., 1998 zo29_225 E32 (group Z)Maiden et al., 1998 Gonococcus zo32_225 Ng F62 Maiden et al., 1998zo33_225 Ng SN4 Chiron SpA fa1090 FA1090 Chiron SpA

An alignment of the sequences generated using PILEUP is shown in FIGS.3A-3C. Stretches of conserved amino acids are evident. The first 74amino acids, for instance, are conserved, although the isoleucine atresidue 51 is not present in 100% of Neisseria. The C-terminal of theprotein consists of 148 conserved amino acids. A similar alignment isshown in FIGS. 13A-13B.

The conserved regions identified in this example confirm that fragmentsof the full-length 225 protein are suitable as multi-specific vaccinesor diagnostic reagents.

Example 4

Example 16 of WO99/57280 discloses the cloning and expression of aNeisserial protein referred to as “235”. Protein and DNA sequences fromserogroup A and B N. meningitidis are disclosed, along with sequencesfrom N. gonorrhoeae.

235 has now been sequenced for a reference population of 31 strains ofNeisseria:

Identification number Strain Reference Group B gnmzq01 NG6/88 Seiler etal., 1996 gnmzq02 BZ198 Seiler et al., 1996 gnmzq03 NG3/88 Seiler etal., 1996 gnmzq04 1000 Seiler et al., 1996 gnmzq05 1000 Seiler et al.,1996 gnmzq07 BZ169 Seiler et al., 1996 gnmzq08 528 Seiler et al., 1996gnmzq09 NGP165 Seiler et al., 1996 gnmzq10 BZ133 Seiler et al., 1996gnmzq11 NGE31 Seiler et al., 1996 gnmzq13 NGE28 Seiler et al., 1996gnmzq14 NGH38 Seiler et al., 1996 gnmzq15 SWZ107 Seiler et al., 1996gnmzq16 NGH15 Seiler et al., 1996 gnmzq17 NGH36 Seiler et al., 1996gnmzq18 BZ232 Seiler et al., 1996 gnmzq19 BZ83 Seiler et al., 1996gnmzq21 MC58 Virji et al., 1992 Group A gnmzq22 205900 Chiron SpAgnmzq23 F6124 Chiron SpA z2491 Z2491 Maiden et al., 1998 Group C gnmzq2490/18311 Chiron SpA gnmzq25 93/4286 Chiron SpA Others gnmzq26 A22 (groupW) Maiden et al., 1998 gnmzq27 E26 (group X) Maiden et al., 1998 gnmzq28860800(group Y) Maiden et al., 1998 gnmzq29 E32 (group Z) Maiden et al.,1998 gnmzq31 N. lactamica Chiron SpA Gonococcus gnmzq32 Ng F62 Maiden etal., 1998 gnmzq33 Ng SN4 Chiron SpA fa1090 FA1090 Dempsey et al. 1991

An alignment of the sequences generated using PILEUP is shown in FIGS.4A-4B. Stretches of conserved amino acids are evident. The protein iswholly conserved, although the serine at residue 168 shows somevariance.

235 was re-sequenced for 35 strains in total, and the sequences werealigned. The results are shown in FIGS. 14A-14B.

Example 5

Example 16 of WO99/57280 discloses the cloning and expression of aNeisserial protein referred to as “287”. Protein and DNA sequences fromserogroup A and B N. meningitidis are disclosed, along with sequencesfrom N. gonorrhoeae.

287 has now been sequenced for a reference population of 6 strains ofNeisseria:

Identification number Strain Reference Group B 287_2 BZ198 Seiler etal.(1996) 287_9 NGP165 Seiler et al.(1996) 287_14 NGH38 Seiler etal.(1996) 287_21 MC58 Virji et al. (1992) Group A z2491 Z2491 Maiden etal.(1998) Gonococcus fa1090 FA1090 Dempsey et al. (1991)

An alignment of the sequences generated using PILEUP is shown in FIG. 5.Stretches of conserved amino acids are evident. The first 42 aminoacids, for instance, are well conserved and a long conserved region canbe seen at the C-terminus.

The conserved regions identified in this example confirm that fragmentsof the full-length 287 protein are suitable as multi-specific vaccinesor diagnostic reagents.

287 was re-sequenced for 35 strains in total (including C1 1, aserogroup C N. meningitidis strain), and the sequences were aligned. Theresults are shown in FIGS. 15A-15E.

Conserved regions of particular interest are:

(SEQ ID NO:222) MFKRSVIAMACI (SEQ ID NO:223) ALSACGGGGGGSPDVKSADT (SEQID NO:224) SKPAAPVV (SEQ ID NO:225) QDMAAVS (SEQ ID NO:226) ENTGNGGAATTD(SEQ ID NO:254) QNDMPQ (SEQ ID NO:274) DGPSQNITLTHCK (SEQ ID NO:255)KSEFE (SEQ ID NO:228) RRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRYLTYGAEKL (SEQ ID NO:256) GGSYAL (SEQ ID NO:229)VQGEPAKGEMLAGTAVYNGEVLHFH (SEQ ID NO:230) GRFAAKVDFGSKSVDGIIDSGDDLHMG(SEQ ID NO:231) QKFKAAIDGNGFKGTWTENGGGDVSG (R/K) FYGPAGEEVAGKYSYRPTDAEKGGFGVFAGKKDRD

Example 6

Example 16 of WO99/57280 discloses the cloning and expression of aNeisserial protein referred to as “519”. Protein and DNA sequences fromserogroup A and B N. meningitidis are disclosed, along with sequencesfrom N. gonorrhoeae.

519 has now been sequenced for a reference population of 22 strains ofNeisseria:

Identification number Strain Source Group B zv01_519 NG6/88 Seiler etal., 1996 zv02_519 BZ198 Seiler et al., 1996 zv03_519ass NG3/88 Seileret al., 1996 zv04_519 297-0 Seiler et al., 1996 zv05_519 1000 Seiler etal., 1996 zv06_519ass BZ147 Seiler et al., 1996 zv07_519 BZ169 Seiler etal., 1996 zv11_519 NGE31 Seiler et al., 1996 zv12_519 NGF26 Seiler etal., 1996 zv18_519 BZ232 Seiler et al., 1996 zv19_519 BZ83 Seiler etal., 1996 zv20_519ass 44/76 Seiler et al., 1996 zv21_519ass MC58 ChironSpA zv96_519 2996 Chiron SpA Group A zv22_519ass 205900 Chiron SpAz2491_519 Z2491 Maiden et al., 1998 Others zv26_519 A22 (group W) Maidenet al., 1998 zv27_519 E26 (group X) Maiden et al., 1998 zv28_519 860800(group Y) Maiden et al., 1998 zv29_519ass E32 (group Z) Maiden et al.,1998 Gonococcus zv32_519 Ng F62 Maiden et al., 1998 fa1090_519 FA1090Chiron SpA

An alignment of the sequences generated using PILEUP is shown in FIGS.6A-6B. Stretches of conserved amino acids are evident, and the proteinshows conservation along its complete length.

519 was re-sequenced for 33 strains in total, and the sequences werealigned. The results are shown in FIGS. 16A-16B.

The conserved regions identified in this example confirm that fragmentsof the full-length 519 protein are suitable as multi-specific vaccinesor diagnostic reagents.

519 was re-sequenced for 33 strains in total, and the sequences werealigned. The results are shown in FIG. 16.

Conserved regions of particular interest are:

(SEQ ID NO:232) MEEFIILL (SEQ ID NO:233)AVAVFGFKSFWIPQQEVHVVERLGRFHRALTAGLNILIPFIDRVAYRHSLKEIPLDVPSQVCITRDNTQLTVDGTTYFQVTDPKLASYGSSNYIMATTQLAQTTLRSVIGRMELDKTFEERDETNSTVV (SEQ ID NO:234)ALDEAAGAWGVKVLRYEIKDLVPPQEILRSMQAQITAEREKRARIAESEGRKTEQINLASGQREAEIQQSEGEAQAAVNASNAEKIARINPAKGEAESLRLVAEANAEANRQIAAALQTQSGADAVNLKIAGQYVTAFKNLAKEDNTRIKPAKVAEIGNPNFRRHEKFSPEAKTAK

Example 7

Example 16 of WO99/57280 discloses the cloning and expression of aNeisserial protein referred to as “919”. Protein and DNA sequences fromserogroup A and B N. meningitidis are disclosed, along with sequencesfrom N. gonorrhoeae.

919 has now been sequenced for a reference population of 35 strains ofNeisseria:

Identification number Strains Source Group B zm01 NG6/88 Seiler et al.,1996 zm02 BZ198 Seiler et al., 1996 zm03 NG3/88 Seiler et al., 1996 zm04297-0 Seiler et al., 1996 zm05 1000 Seiler et al., 1996 zm06 BZ147Seiler et al., 1996 zm07 BZ169 Seiler et al., 1996 zm08n 528 Seiler etal., 1996 zm09 NGP165 Seiler et al., 1996 zm10 BZ133 Seiler et al., 1996zm11asbc NGE31 Seiler et al., 1996 zm12 NGF26 Seiler et al., 1996 zm13NGE28 Seiler et al., 1996 zm14 NGH38 Seiler et al., 1996 zm15 SWZ107Seiler et al., 1996 zm16 NGH15 Seiler et al., 1996 zm17 NGH36 Seiler etal., 1996 zm18 BZ232 Seiler et al., 1996 zm19 BZ83 Seiler et al., 1996zm20 44/76 Seiler et al., 1996 zm21 MC58 Chiron SpA zm96 2996 Chiron SpAGroup A zm22 205900 Chiron SpA zm23asbc F6124 Chiron SpA z2491 Z2491Maiden et al., 1998 Group C zm24 90/18311 Chiron SpA zm25 93/4286 ChironSpA Others zm26 A22 (group W) Maiden et al., 1998 zm27bc E26 (group X)Maiden et al., 1998 zm28 860800 (group Y) Maiden et al., 1998 zm29asbcE32 (group Z) Maiden et al., 1998 zm31asbc N. lactamica Chiron SpAGonococcus zm32asbc Ng F62 Maiden et al., 1998 zm33asbc Ng SN4 ChironSpA fa1090 FA1090 Chiron SpA

An alignment of the sequences generated using PILEUP is shown in FIGS.7A-7D. Another alignment is shown in FIGS. 18A-18C. Stretches ofconserved amino acids are evident. The protein shows almost completeconservation.

The conserved regions identified in this example confirm that fragmentsof the full-length 919 protein are suitable as multi-specific vaccinesor diagnostic reagents.

Conserved regions of particular interest are:

(SEQ ID NO:235) MKKYLFRAAL (SEQ ID NO:236)GIAAAILAACQSKSIQTFPQPDTSVINGPDRPVGIPDPAGTTV (G/A)GGGAVYTVVPHLSLPHWAAQDFAKSLQSFRLGCANLKNRQGWQDVCAQAFQTPVHSFQAKQFFERYFTPWQVAGNGSLAGTVTGYYEPVLKGDDRRTAQARFPIYGIPDDFISVPLPAGLRSGKALVRIRQTGKNSGTIDN (SEQ ID NO:237) GGTHTADLS (SEQID NO:238) FPITARTTAIKGRFEGSRFLPYHTRNQINGGALDGKAPILGYAEDPVELFFMHIQGSGRLKTPSGKYIRIGYADKNEHPYVSIG (R/K) YMADKGYLK LGQTSMQGIK (SEQ IDNO:239) YMRQNPQRLAEVLGQNPSYIFFREL (SEQ ID NO:240)NDGPVGALGTPLMGEYAGAVDRHYITLGAPLFVATAHPVTRKALNRLIMAQDTGSAIKGAVRVDYFWGYGDEAGELAGKQKTTGYVWQLLPNGMKPEYRP

Example 8

Example 55 of WO99/23578 discloses the cloning and expression of aNeisserial protein referred to as “ORF46”. Protein and DNA sequencesfrom serogroups A and B N. meningitidis are disclosed, along withsequences from N. gonorrhoeae.

Full-length ORF46 has been sequenced for a reference population of 6strains of serogroup B. An alignment of these sequences is shown inFIGS. 12A-12B, from which stretches of conserved amino acids areevident.

Conserved regions of particular interest are:

(SEQ ID NO:241) RKISLILSILAVCLPMHAHASDLANDSFTRQVLDRQHFEPDGKYHLFGSRGELAERSGHIGLG (SEQ ID NO:242)IQSHQLGNLMIQQAAIKGNIGYIVRFSDHGHEVHSPFDNHASHSDSDEAGSPVDGFSLYRIHWDGYEHHPADGYDGPQGGGYPAPKGARDIYSYDIKGVAQNIRLNLTDNRSTGQRLADRFHNAG (SEQ ID NO:243)MLTQGVGDGFKRATRYSPELDRSGNAAEAFNGTADIVKNIIGAAGEIVGAGDAVQGISEGSNIAVMHGLGLLSTENKNARINDLADNAQLKDYAAAAIRD WAVQNPNAAQGIEAVSNIF(SEQ ID NO:244) IPIKGIGAVRGKYGLGGITAHP (V/I) KRSQMGEIALPKGKSAVS (SEQ IDNO:245) NFADAAYAKYPSPYHSRNIRSNLEQRYGKENITSSTVPPSNGKNVKLANKRHPKTKVPFDGKGFPNFEKDVKY

The conserved regions in ORF46 confirm that fragments of this proteinare suitable as multi-specific vaccines or diagnostic reagents.

Example 9

WO99/57280 discloses the cloning and expression of a Neisserial proteinreferred to as “726”. Protein and DNA sequences from serogroups A and BN. meningitidis are disclosed.

726 has been sequenced for a reference population of 7 N. meningitidisstrains in serogroups A, B and C. An alignment of these sequences isshown in FIG. 17, from which stretches of conserved amino acids areevident.

Conserved regions of particular interest are:

(SEQ ID NO:246) IYFKNGFYDDTLG (SEQ ID NO:247)IPEGAVAVRAEEYAALLAGQAQGGQIAADSDGRPVLTPPRPS (D/E) YHEWDGKKW (SEQ IDNO:248) AAAAARFAEQKTATAFRLA (SEQ ID NO:249)KADELKNSLLAGYPQVETDSFYRQEKEALARQADNNAPTPMLAQIAAARG VELDVLTEKV (I/V)EKSARLAVAAGAIIGKRQQLEDKLN (SEQ ID NO:250) IETAPGLDALEKEIEEWT

The conserved regions in 726 confirm that fragments of this protein aresuitable as multi-specific vaccines or diagnostic reagents.

Example 10

WO99/57280 discloses the cloning and expression of a Neisserial proteinreferred to as “953”. Protein and DNA sequences from serogroups A and BN. meningitidis are disclosed, along with sequences from N. gonorrhoeae.

953 has been sequenced for a reference population of 8 strains of N.meningitidis serogroups A, B and C. An alignment of these sequences isshown in FIG. 19, from which stretches of conserved amino acids areevident. The protein is well-conserved.

Conserved regions of particular interest are:

(SEQ ID NO:251) MKKIIFAALAAAAVGTASAATYKVDEYHANARFAIDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITIP (I/V) ANLQSGSQHFTDHLKSADIFDAAQYPDTRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPM (SEQ ID NO:252)ATYKVDEYHANARFATDHFNTSTNVGGFYGLTGSVEFDQAKRDGKIDITI P (I/V)ANLQSGSQHFTDHLKSADIFDAAQYPDIRFVSTKFNFNGKKLVSVDGNLTMHGKTAPVKLKAEKFNCYQSPM (SEQ ID NO:253) KTEVCGGDFSTTIDRTKWG(M/V) DYLVNVGMTKSVRIDIQIEAAKQ

The conserved regions in 953 confirm that fragments of this protein aresuitable as multi-specific vaccines or diagnostic reagents.

Phylogenetic Tree

FIG. 8 is a dendrogram showing the genetic relationship among 107 N.meningitidis strains, based on MLST analysis of six gene fragments[adapted from Maiden et al. (1998) PNAS USA 95:3140]. The dendrogram canbe used to select strains representative of meningococcus serogroup B(arrows). Five additional strains, for which genetic assignment tohypervirulent lineages has been independently determined by Wang et al.[J. Infect. Dis (1993) 167:1320], Seiler et al. [Mol. Microbiol. (1996)19:841], and Virji et al. [Mol. Microbiol. (1992) 6:1271] aresuperimposed on the dendrogram and indicated by asterisks. In additionto the 22 strains of MenB, three strains of MenA, two strains of MenC,and one strain each of Men Y, X, Z and W135 were used. These areindicated by bold letters before the name. Where phylogenetic data werenot available, the strains are shown outside the tree. The hypervirulentstrains ET-5, ET-37 and IV-1 are indicated.

Sequence Variability

FIG. 9 a is a schematic representation of amino acid sequencevariability with N. meningitidis for proteins 225, 235, 287, 519, 919,ORF4 and ORF40. The horizontal axis represents the sequence of MC58.Amino acid differences within MenB strains are indicated by verticallines above the horizontal axis; differences within serogroups A, C, Y,X, Z and W135 are indicated by lines below the axis. The height of thevertical lines represents the number of strains with amino aciddifferences. Peaks thus show variable regions. The bard below 225 and287 represent sequence segments that are missing from some strains.

FIG. 9 b is a dendrogram of N. meningitidis strains obtained using thesame 7 proteins. The phylogenetic analysis based on these genes provideda dendrogram which clusters the hypervirulent strains in agreement withFIG. 8. Bars indicate strains which cluster with 100% bootstrap supportin agreement with MLST analysis. Numbers at the base of each node arebootstrap scores (only those >80% are reported). Gene sequences fromdifferent strains were aligned with the program PILEUP from the GCGpackage. The phylogenetic analysis was performed using theneighbour-joining algorithm [Saitou & Nei (1987) Mol. Biol. Evol. 4:406]as implemented in the NEIGHBOR program of the PHYLIP package. Pairwisedistances were calculated using the Kimura-two parameter [Kimura (1980)J. Mol. Evol. 16:111] on the 31 N. meningitidis strains. The N-terminalregion of ORF40, the entire 287, and the tandem repeats of 225 wereexcluded from the analysis. A total of 1000 bootstrap replicates wereallowed to evaluate the level of support. The clustering of thehypervirulent strains was confirmed by maximum parsimony analysis.

Western Blots

Western blots Antigens ORF4, 225, 235, 519 and 919 were analysed byWestern blot for various strains. The results are shown in FIGS.20A-20E. In the case of 225, the blot shows fragments of different sizesin the different strains, with arrows indicating the band of correctsize. 225 contains regions of deletion and insertion of a defined repeatand the size of the fragments on the blots matches the gene variabilitydata.

The strains used for FIGS. 20A-20E are as follows:

N. meningitidis Serogroup B:

 1 = NG6/88  2 = BZ198  3 = NG3/88  4 = 297-0  5 = 1000  6 = BZ147  7 =BZ169  8 = 528  9 = NGP165 10 = BZ133 11 = NGE31 12 = NGF26 13 = NGE2814 = NGH38 15 = SWZ107 16 = NGH15 17 = NGH36 18 = BZ232 19 = BZ83 20 =44/76 21 = MC58 96 = 2996

N. meningitidis Serogroup A:

-   -   22=205900 23=F6124

N. meningitidis Serogroup C:

-   -   24=90/18311 25=93/4286

Other N. meningitidis

-   -   26=A22 (serogroup W)    -   27=E26 (serogroup X)    -   28=860800 (serogroup Y)    -   29=E32 (serogroup Z)

Other Neisseria

-   -   30=N. cinerea    -   L17=N. lactamica    -   L19=N. lactamica    -   31=N. gonorrhoeae F62    -   32=N. gonorrhoeae SN4

It will be appreciated that the invention has been described by means ofexample only, and that modifications may be made whilst remaining withinthe spirit and scope of the invention.

1-15. (canceled) 16: An isolated protein fragment comprising the aminoacid sequence (SEQ ID NO:222) MFKRSVIAMACI, (SEQ ID NO:223)ALSACGGGGGGSPDVKSADT, (SEQ ID NO:224) SKPAAPVV, (SEQ ID NO:225) QDMAAVS,(SEQ ID NO:226) ENTGNGGAATTD, (SEQ ID NO:254) QNDMPQ, (SEQ ID NO:227)DGPSQNITLTHCK, (SEQ ID NO:255) KSEFE, (SEQ ID NO:228)RRSARSRRSLPAEMPLIPVNQADTLIVDGEAVSLTGHSGNIFAPEGNYRY LTYGAEKL, (SEQ IDNO:256) GGSYAL, (SEQ ID NO:229) VQGEPAKGEMLAGTAVYNGEVLHFH, (SEQ IDNO:230) GRFAAKVDFGSKSVDGIIDSGDDLHMG, or (SEQ ID NO:231)QKFKAAIDGNGFKGTWTENGGGDVSG (R/K) FYGPAGEEVAGKYSYRP TDAEKGGFGVFAGKKDRD,

provided that said protein fragment is not full length ORF287. 17: Theisolated protein fragment of claim 16 consisting of the amino acidsequence of SEQ ID NO:227. 18: The isolated protein fragment of claim 16consisting of the amino acid sequence of SEQ ID NO:228. 19: The isolatedprotein fragment of claim 16 consisting of the amino acid sequence ofSEQ ID NO:230. 20: The isolated protein fragment of claim 16 furthercomprising an adjuvant. 21: A method for treating or preventinginfection due to Neisserial bacteria, said method comprisingadministering a protein according to claim 16 to a subject in needthereof. 22: A method of diagnosing an infection due to Neisserialbacteria, said method comprising contacting a biological sample with aprotein according to claim 16, under conditions whereby anantibody/antigen complex is formed if antibodies to a Neisserialbacterium is present in the biological sample, and detecting saidcomplex.